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United States Patent 5,708,158
Hoey January 13, 1998
Nuclear factors and binding assays

Abstract

The invention provides methods and compositions for identifying pharmacological agents useful in the diagnosis or treatment of disease associated with the expression of a gene modulated by a transcription complex containing at least a human nuclear factor of activated T-cells (hNFAT). The materials include a family of hNFAT proteins, active fragments thereof, and nucleic acids encoding them. The methods are particularly suited to high-throughput screening where one or more steps are performed by a computer controlled electromechanical robot comprising an axial rotatable arm.

Inventors: Hoey; Timothy (Woodside, CA)
Assignee: Tularik Inc. (South San Francisco, CA)
Appl. No.: 818823
Filed: March 14, 1997

Current U.S. Class: 536/23.5; 536/23.1
Intern'l Class: C12N 015/12; C12N 015/11
Field of Search: 536/23.1,23.5

References Cited

Other References

Li et al, PNAS (USA), Sep., 1991, vol. 88: pp. 7739-7743.
Primary Examiner: Elliott; George C.
Assistant Examiner: McKelvey; Terry A.
Attorney, Agent or Firm: Osman; Richard Aron
Parent Case Text


This is a division of application Ser. No. 08/396,479 filed Mar. 02, 1995, now U.S. Pat. No. 5,612,455 which is a continuation-in-part application of Ser. No. 08/270,653 filed on Jul. 5, 1994, now abandoned.
Claims


What is claimed is:

1. An isolated nucleic acid encoding a human nuclear factor of activated T-cells, hNFAT, protein comprising hNFATp.sub.1 (SEQ ID NO:2), hNFATp.sub.2 (SEQ ID NO:2, residues 220-921), hNFAT3 (SEQ ID NO:6), hNFAT4b (SEQ ID NO:10) or hNFAT4c (SEQ ID NO:12).

2. The isolated nucleic acid of claim 1, wherein said protein comprises hNFATp.sub.1 (SEQ ID NO:2).

3. The isolated nucleic acid of claim 1, wherein said protein comprises hNFATp.sub.2 (SEQ ID NO:2, residues 220-921).

4. The isolated nucleic acid of claim 1, wherein said protein comprises hNFAT3 (SEQ ID NO:6).

5. The isolated nucleic acid of claim 1, wherein said protein comprises hNFAT4b (SEQ ID NO:10).

6. The isolated nucleic acid of claim 1, wherein said protein comprises hNFAT4c (SEQ ID NO:12).

7. An isolated nucleic acid encoding an hNFAT protein, said nucleic acid comprising SEQ ID NO:1, SEQ ID NO:5, SEQ ID NQ:9, SEQ ID NO:11 or nucleotides 1-356 and 868-3478 of SEQ ID NO:1.

8. The isolated nucleic acid of claim 7, wherein said nucleic acid comprises SEQ ID NO:1.

9. The isolated nucleic acid of claim 7, wherein said nucleic acid comprises nucleotides 1-356 and 868-3478 of SEQ ID NO:1.

10. The isolated nucleic acid of claim 7, wherein said nucleic acid comprises SEQ ID NO:5.

11. The isolated nucleic acid of claim 7, wherein said nucleic acid comprises SEQ ID NO:9.

12. The isolated nucleic acid of claim 7, wherein said nucleic acid comprises SEQ ID NO:1.
Description


INTRODUCTION

1. Field of the Invention

The field of this invention is human transcription factors of activated T-cells.

2. Background

Identifying and developing new pharmaceuticals is a multibillion dollar industry in the U.S. alone. Gene specific transcription factors provide a promising class of targets for novel therapeutics directed to these and other human diseases. Urgently needed are efficient methods of identifying pharmacological agents or drugs which are active at the level of gene transcription. If amenable to automated, cost-effective, high throughput drug screening, such methods would have immediate application in a broad range of domestic and international pharmaceutical and biotechnology drug development programs.

Immunosuppression is therapeutically desirable in a wide variety of circumstances including transplantation, allergy and other forms of hypersensitivity, autoimmunity, etc. Cyclosporin, a widely used drug for effecting immunosuppression, is believed to act by inhibiting a calcineurin, a phosphatase which activates certain nuclear factors of activated T-cells (NFATs). However, because of side effects and toxicity, clinical indications of cyclosporin (and the more recently developed FK506) are limited.

Accordingly, it is desired to identify agents which more specifically interfere with the function of hNFATs. Unfortunately, the reagents necessary for the development of high-throughput screening assays for such therapeutics are unavailable.

3. Relevant Literature

Nolan (Jun. 17, 1994) Cell 77, 1-20 provides a recent review and commentary on molecular interactions of hNFAT proteins. Northrop et at. (Jun. 9, 1994) Nature 369, 497-502 report the cloning of a cDNA encoding human NFATc. McCaffrey et al. (Oct. 29, 1993) Science 262, 750-754 report the cloning of a fragment of a gene encoding a murine NFATp.sub.1.

SUMMARY OF THE INVENTION

The invention provides methods and compositions for identifying lead compounds and pharmacological agents useful in the diagnosis or treatment of disease associated with the expression of one or more genes modulated by a transcription complex containing a human nuclear factor of activated T-cells (hNFAT). Several forms of hNFAT are provided including hNFATs designated hNFATp.sub.1, hNFATp.sub.2, hNFATc, hNFAT3, hNFAT4a, hNFAT4b and hNFAT4c. The invention also provides isolated nucleic acid encoding the subject hNFATs, vectors and cells comprising such nucleic acids, and methods of recombinantly producing polypeptides comprising hNFAT. The invention also provides hNFAT-specific binding reagents such as hNFAT-specific antibodies.

Methods using the disclosed hNFATs in drug development programs involve combining a selected hNFAT with a natural intracellular hNFAT binding target and a candidate pharmacological agent. Natural intracellular binding targets include transcription factors, such as AP1 proteins and nucleic acids encoding a hNFAT binding sequence. The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the hNFAT selectively binds the target. Then the presence or absence of selective binding between the hNFAT and target is detected. A wide variety of alternative embodiments of the general methods using hNFATs are disclosed. The methods are particularly suited to high-throughput screening where one or more steps are performed by a computer controlled electromechanical robot comprising an axial rotatable arm and the solid substrate is a portion of a well of a microtiter plate. 

    ______________________________________
                   hNFAT SEQ ID NOS:
    ______________________________________
    hNFATp.sub.1
               cDNA      SEQ ID NO: 1
    hNFATp.sub.1
               protein   SEQ ID NO: 2
    hNFATp.sub.2
               cDNA      SEQ ID NO: 1, bases 1-356
                         and 868-3478
    hNFATp.sub.2
               protein   SEQ ID NO: 2, residues 220-921
    hNFATc     cDNA      SEQ ID NO: 3
    hNFATc     protein   SEQ ID NO: 4
    hNFAT3     cDNA      SEQ ID NO: 5
    hNFAT3     protein   SEQ ID NO: 6
    hNFAT4a    cDNA      SEQ ID NO: 7
    hNFAT4a    protein   SEQ ID NO: 8
    hNFAT4b    cDNA      SEQ ID NO: 9
    hNFAT4b    protein   SEQ ID NO: 10
    hNFAT4c    cDNA      SEQ ID NO: 11
    hNFAT4c    protein   SEQ ID NO: 12
    ______________________________________


DETAILED DESCRIPTION OF THE INVENTION

The invention provides methods and compositions relating to human NFATs. The subject hNFATs include regulators of cytokine gene expression that modulate immune system function. As such, hNFATs and HNFAT-encoding nucleic acids provide important targets for therapeutic intervention.

hNFATs derive from human cells, comprise invariant hNFAT rel domain peptides (see, Table 1)and share at least 50% pair-wise rel sequence identity with each of the disclosed hNFAT sequences. Invariant hNFAT rel domain peptides include from the N-terminal end of the rel domain, HHRAHYETEGSRGAVKA (SEQ ID NO:2, residues 419-435), PHAFYQVHRITGK (SEQ ID NO:2, residues 470-482), IDCAGILKLRN (SEQ ID NO:2, residues 513-523), DIELRKGETDIGRKNTRVRLVFRVHX,P (SEQ ID NO: 13), and PX.sub.2 ECSQRSAX.sub.3 ELP (SEQ ID NO: 14), where each X.sub.1 and X.sub.2 is hydrophobic residue such as valine or isoleucine, and X.sub.3 is any residue, but preferably glutamine or histidine. 

                                      TABLE 1
    __________________________________________________________________________
    hNFAT rel domains
    NFATp (SEQ ID NO: 2, residues 388-678)
    NFATc (SEQ ID NO: 4, residues 406-697)
    NFAT3 (SEQ ID NO: 6, residues 397-686)
    NFAT4b/c (SEQ ID NO: 10, resiudes 411-700)
    __________________________________________________________________________
    NFATp
         IPVTASLPPLEWPLSSQSGSYELRIEVQPKPHHRAHYETEGSRGAVKAPT
                                            50
    NFATc
         SYMSPTLPALDWQLPSHSGPYELRIEVQPKSHHRAHYETEGSRGAVKASA
                                            50
    NFAT3
         IFRTSALPPLDWPLPSQYEQLELRIEVQPRAHHRAHYETEGSRGAVKAAP
                                            50
    NFAT4b/c
         IFRTSSLPPLDWPLPAHFGQCELKIEVQPKTHHRAHYETEGSRGAVKAST
                                            50
    NFATp
         GGHPVVQLHGYMENKPLGLQIFIGTADERILKPHAFYQVHRITGKTVTTT
                                            100
    NFATc
         GGHPIVQLHGYLENEPLMLQLFIGTADDRLLRPHAFYQVHRITGKTVSTT
                                            100
    NFAT3
         GGHPVVKLLGYS-EKPLTLQMFIGTADERNLRPHAFYQVHRITGKMVATA
                                            99
    NFAT4b/c
         GGHPVVKLLGYN-EKPINLQMFIGTADDRYLRPHAFYQVHRITGKTVATA
                                            99
    NFATp
         SYEKIVGNTKVLEIPLEPKNNMRATIDCAGILKLRNADIELRKGETDIGR
                                            150
    NFATc
         SHEAILSNTKVLEIPLLPENSMRAVIDCAGILKLRNSDIELRKGETDIGR
                                            150
    NFAT3
         SYEAVVSGTKVLEMTLLPENNMAANIDCAGILKLRNSDIELRKGETDIGR
                                            149
    NFAT4b/c
         SQEIIIASTKVLEIPLLPENNMSASIDCAGILKLRNSDIELRKGETDIGR
                                            149
    NFATp
         KNTRVRLVFRVHIPESSGRIVSLQTASNPIECSQRSAHELPMVERQDTDS
                                            200
    NFATc
         KNTRVRLVFRVHVPQPSGRTLSLQVASNPIECSQRSAQELPLVEKQSTDS
                                            200
    NFAT3
         KNTRVRLVFRVHVPQGGGKVVSVQAASVPIECSQRSAQELPQVEAYSPSA
                                            199
    NFAT4b/c
         KNTRVRLVFRVHIPQPSGKVLSLQIASIPVECSQRSAQELPHIEKYSINS
                                            199
    NFATp
         CLVYGGQQMILTGQNFTSESKVVFTEKTTDGQQIWEMEATVDKDKSQPNM
                                            250
    NFATc
         YPVVGGKKMVLSGHNFLQDSKVIFVEKAPDGHHVWEMEAKTDRDLCKPNS
                                            250
    NFAT3
         CSVRGGEELVLTGSNFLPDSKVVFIERGPDGKLQWEEEATVNRLQSNEVT
                                            249
    NFAT4b/c
         CSVNGGHEMVVTGSNFLPESKIIFLEKGQDGRPQWEVEGKIIREKCQGAH
                                            249
    NFATp
         LFVEIPEYRNKHIRTPVKVNFYVINGKRKRSQPQHFTYHPV
                                            291
    NFATc
         LVVEIPPFRNQRITSPVHVSFYVCNGKRKRSQYQRFTYLPA
                                            291
    NFAT3
         LTLTVPEYSNKRVSRPVQVYFYVSNGRRKRSPTQSFRFLPV
                                            290
    NFAT4b/c
         IVLEVPPYHNPAVTAAVQVHFYLCNGKRKKSQSQRFTYTPV
                                            290
    __________________________________________________________________________


In addition to the shared rel domains, some hNFATs have smaller regions of sequence similarity on the terminal side of the rel domains. For example, the amino terminal regions of hNFAT 4a, 4b and 4c and hNFATc have several regions of similarity (Table 2). The two largest regions (designated regions A and B in Table 2) contain 23 of 41 and 24 of 45 identical amino acids between the two proteins. hNFATp and hNFAT3 also have similarity to other hNFAT proteins in this region (Table 2). The homology between hNFAT3 and hNFAT 4a, 4b and 4c extends about 25 amino acids upstream of the rel region (designated region C in Table 2). 

                                      TABLE 2
    __________________________________________________________________________
    hNFAT regions 5' to the rel domain
    __________________________________________________________________________
        NFATc
             PSTATLSLPSLEAYRDPS-CLSPASSLSSRSCNSEASSYES
                                          195
        NFAT4a
             PSRDHLYLPLEPSYRESSLSPSPASSISSRSWFSDASSCES
                                          189
    A   NFATc (SEQ ID NO: 4, residues 152-191)
        NFAT4a (SEQ ID NO: 8, residues 144-184)
        NFATc
             SPQHSPSTSPRASVTEESWLGAR-----SSRPASPCNKRKYSLNG
                                          272
        NFAT4a
             SPRQSPCHSPRSSVTDENWLSPRPASGPSSRPTSPCGKRRSSAEV
                                          281
        NFATc (SEQ ID NO: 4, residues 233-272)
        NFAT4a (SEQ ID NO: 8, residues 236-281)
        NFATc
             SSRPASPCNKRKYSLNG            272
        NFAT3
             SPRPASPCGKRRYSSSG            275
    B   NFATc (SEQ ID NO: 4, residues 256-272)
        NFAT3 (SEQ ID NO: 6, residues 259-275)
        NFATc
             SPQHSPSTSPRASVTEESWLGARSSRP  272
        NFATp
             SPRTSPIMSPRTSLAEDSCLGRHSPVP  239
        NFATc (SEQ ID NO: 4, residues 233-259)
        NFATp (SEQ ID NO: 2, residues 213-239)
        NFAT3
             RKEVAGMDYLAVPSPLAWSKARIGGHSP 396
        NFAT4a
             KKDSCGDQFLSVPSPFTWSKPKPG-HTP 410
    C   NFAT3 (SEQ ID NO: 6, residues 369-396)
        NFAT4a (SEQ ID NO: 8, residues 384-410)
    __________________________________________________________________________


Nucleic acids encoding hNFATs may be isolated from human cells by screening cDNA libraries for human immune cells with probes or PCR primers derived from the disclosed hNFAT genes. In addition to the invariant hNFAT rel sequences and the 50% pair-wise rel domain identity, cDNAs of hNFAT transcripts typically share substantially overall sequence identity with one or more of the disclosed hNFAT sequences.

The subject hNFAT fragments have one or more hNFAT-specific binding affinities, including the ability to specifically bind at least one natural human intracellular hNFAT-specific binding target or a hNFAT-specific binding agent such as a hNFAT-specific antibody or a hNFAT-specific binding agent identified in assays such as described below. Accordingly, the specificity of hNFAT fragment specific binding agents is confirmed by ensuring non-cross-reactivity with other NFATs. Furthermore, preferred hNFAT fragments are capable of eliciting an antibody capable of specifically binding an hNFAT. Methods for making immunogenic peptides through the use of conjugates, adjuvants, etc. and methods for eliciting antibodies, e.g. immunizing rabbits, are well known.

Exemplary natural intracellular binding targets include nucleic acids which comprise one or more hNFAT DNA binding sites. Functional hNFAT binding sites have been found in the promoters or enhancers of several different cytokine genes including IL-2, IL-4, IL-3, GM-CSF, and TNF-a and are often located next to AP-1 binding sites, which are recognized by members of the fos and jun families of transcription factors. Typically, the AP-1 binding sites adjacent to hNFAT sites are low affinity sites, and AP-1 proteins cannot bind them independently. However, many NF-AT and AP-1 protein combinations are capable of cooperatively binding to DNA. Furthermore, cell-type specificity of cytokine gene transcription is often controlled, at least in part, by the combinations of hNFAT and AP-1 proteins present in those cells. For example, there are different classes of T cells that secrete different sets of cytokines: e.g. TH1 cells produce IL-2 and IFN-g, while TH2 cells produce IL-4, IL-5, and IL-6. hNFAT binding sites are involved in the regulation of both TH1 and TH2 cytokines. Further, differential expression of the cytokine gene in T cell subsets is controlled the combinatorial interactions of hNFAT and AP-1 proteins.

In addition to DNA binding sites and other transcription factors such as AP1, other natural intracellular binding targets include cytoplasmic proteins such as ankyrin repeat containing hNFAT inhibitors, protein serine/threonine kinases, etc., and fragments of such targets which are capable of hNFAT-specific binding. Other natural hNFAT binding targets are readily identified by screening cells, membranes and cellular extracts and fractions with the disclosed materials and methods and by other methods known in the art. For example, two-hybrid screening using hNFAT fragments are used to identify intracellular targets which specifically bind such fragments. Preferred hNFAT fragments retain the ability to specifically bind at least one of an hNFAT DNA binding site and can preferably cooperatively bind with AP 1. Convenient ways to verify the ability of a given hNFAT fragment to specifically bind such targets include in vitro labelled binding assays such as described below, and EMSAs.

A wide variety of molecular and biochemical methods are available for generating and expressing hNFAT fragments, see e.g. Molecular Cloning, A Laboratory Manual (2nd Ed., Sambrook, Fritsch and Maniatis, Cold Spring Harbor), Current Protocols in Molecular Biology (Eds. Aufubel, Brent, Kingston, More, Feidman, Smith and Stuhl, Greene Publ. Assoc., Wiley-Interscience, New York, N.Y., 1992) or that are otherwise known in the art. For example, hNFAT or fragments thereof may be obtained by chemical synthesis, expression in bacteria such as E. coli and eukaryotes such as yeast or vaccinia or baculovirus-based expression systems, etc., depending on the size, nature and quantity of the hNFAT or fragment. The subject hNFAT fragments are of length sufficient to provide a novel peptide. As used herein, such peptides are at least 5, usually at least about 6, more usually at least about 8, most usually at least about 10 amino acids. hNFAT fragments may be present in a free state or bound to other components such as blocking groups to chemically insulate reactive groups (e.g. amines, carboxyls, etc.) of the peptide, fusion peptides or polypeptides (i.e. the peptide may be present as a portion of a larger polypeptide), etc.

The subject hNFAT fragments maintain binding affinity of not less than six, preferably not less than four, more preferably not less than two orders of magnitude less than the binding equilibrium constant of a full-length native hNFATto the binding target under similar conditions. Particular hNFAT fragments or deletion mutants are shown to function in a dominant-negative fashion. Such fragments provide therapeutic agents, e.g. when delivered by intracellular immunization--transfection of susceptible cells with nucleic acids encoding such mutants.

The claimed hNFAT and hNFAT fragments are isolated, partially pure or pure and are typically recombinantly produced. As used herein, an "isolated" peptide is unaccompanied by at least some of the material with which it is associated in its natural state and constitutes at least about 0.5%, preferably at least about 2%, and more preferably at least about 5% by weight of the total protein (including peptide) in a given sample; a partially pure peptide constitutes at least about 10% , preferably at least about 30%, and more preferably at least about 60% by weight of the total protein in a given sample; and a pure peptide constitutes at least about 70% , preferably at least about 90%, and more preferably at least about 95 % by weight of the total protein in a given sample.

Preferred hNFAT fragments comprise at least a functional portion of the rel domain. There are several different biochemical functions that are mediated by the rel and hNFAT rel-similarity domains: DNA binding, dimerization, interaction with B-zip proteins, interaction with inhibitor proteins, and nuclear localization. Other rel family proteins have been shown to physically interact with AP-1 (fos and jun) proteins (Stein et al., EMBO J. 12, 1993). The rel homology domain is necessary for this interaction and the B-zip region of the AP-1 proteins is involved in this protein-protein interaction. The specificity in the ability of hNFAT and AP-1 family members to interact is related to the tissue specific and cell type specific regulation of gene expression governed by these proteins. The rel and rel-similarity domains also interact with members of the I-kB family of inhibitor proteins including I-kB-like ankyrin repeat proteins (reviewed in Beg and Baldwin, Genes and Dev., 1993). The C-terminal half or the rel domain is involved the interaction with I-kB. There are 5 related I-kB-like proteins which are characterized by having multiple copies of a 33 amino acid sequence motif called the ankyrin repeat.

The invention provides hNFAT-specific binding agents, methods of identifying and making such agents, and their use in diagnosis, therapy and pharmaceutical development. For example, hNFAT-specific agents are useful in a variety of diagnostic applications, especially where disease or disease prognosis is associated with immune disfunction resulting from improper expression of hNFAT. Novel hNFAT-specific binding agents include hNFAT-specific antibodies and other natural intracellular binding agents identified with assays such as one- and two-hybrid screens; non-natural intracellular binding agents identified in screens of chemical libraries, etc.

Generally, hNFAT-specificity of the binding target is shown by binding equilibrium constants. Such targets are capable of selectively binding a hNFAT, i.e. with an equilibrium constant at least about 10.sup.4 M.sup.-1, preferably at least about 10.sup.6 M.sup.-1, more preferably at least about 10.sup.8 M.sup.-1. A wide variety of cell-based and cell-free assays may be used to demonstrate hNFAT-specific binding. Cell based assays include one and two-hybrid screens, mediating or competitively inhibiting hNFAT-mediated transcription, etc. Preferred are rapid in vitro, cell-free assays such as mediating or inhibiting hNFAT-protein (e.g. hNFAT-AP1 binding), hNFAT-nucleic acid binding, immunoassays, etc. Other useful screening assays for hNFAT/hNFAT fragment-target binding include fluorescence resonance energy transfer (FRET), electrophoretic mobility shift analysis (EMSA), etc.

The invention also provides nucleic acids encoding the subject hNFAT and hNFAT fragments, which nucleic acids may be part of hNFAT-expression vectors and may be incorporated into recombinant cells for expression and screening, transgenic animals for functional studies (e.g. the efficacy of candidate drugs for disease associated with expression of a hNFAT), etc. In addition, the invention provides nucleic acids sharing substantial sequence similarity with that of one or more wild-type hNFAT nucleic acids. Substantially identical or homologous nucleic acid sequences hybridize to their respective complements under high stringency conditions, for example, at 55.degree. C. and hybridization buffer comprising 50% formamide in 0.9M saline/0.09M sodium citrate (SSC) buffer and remain bound when subject to washing at 55.degree. C. with the SSC/formamide buffer. Where the sequences diverge, the differences are preferably silent, i.e. or a nucleotide change providing a redundant codon, or conservative, i.e. a nucleotide change providing a conservative amino acid substitution.

The subject nucleic acids find a wide variety of applications including use as hybridization probes, PCR primers, therapeutic nucleic acids, etc. for use in detecting the presence of hNFAT genes and gene transcripts, for detecting or amplifying nucleic acids with substantial sequence similarity such as hNFAT homology and structural analogs, and for gene therapy applications. Given the subject probes, materials and methods for probing cDNA and genetic libraries and recovering homology are known in the art. Preferred libraries are derived from human immune cells, especially cDNA libraries from differentiated and activated human lymphoid cells. In one application, the subject nucleic acids find use as hybridization probes for identifying hNFAT cDNA homologs with substantial sequence similarity. These homologs in turn provide additional hNFATs and hNFAT fragments for use in binding assays and therapy as described herein. hNFAT encoding nucleic acids also find applications in gene therapy. For example, nucleic acids encoding dominant-negative hNFAT mutants are cloned into a virus and the virus used to transfect and confer disease resistance to the transfected cells.

Therapeutic hNFAT nucleic acids are used to modulate, usually reduce, cellular expression or intracellular concentration or availability of active hNFAT. These nucleic acids are typically antisense: single-stranded sequences comprising complements of the disclosed hNFAT nucleic acids. Antisense modulation of hNFAT expression may employ hNFAT antisense nucleic acids operably linked to gene regulatory sequences. Cell are transfected with a vector comprising an hNFAT sequence with a promoter sequence oriented such that transcription of the gene yields an antisense transcript capable of binding to endogenous hNFAT encoding mRNA. Transcription of the antisense nucleic acid may be constitutive or inducible and the vector may provide for stable extrachromosomal maintenance or integration. Alternatively, single-stranded antisense nucleic acids that bind to genomic DNA or mRNA encoding a hNFAT or hNFAT fragment may be administered to the target cell, in or temporarily isolated from a host, at a concentration that results in a substantial reduction in hNFAT expression. For gene therapy involving the transfusion of hNFAT transfected cells, administration will depend on a number of variables that are ascertained empirically. For example, the number of cells will vary depending on the stability of the transfused cells. Transfusion media is typically a buffered saline solution or other pharmacologically acceptable solution. Similarly the amount of other administered compositions, e.g. transfected nucleic acid, protein, etc., will depend on the manner of administration, purpose of the therapy, and the like.

The subject nucleic acids are often recombinant, meaning they comprise a sequence joined to a nucleotide other than that which it is joined to on a natural chromosome. An isolated nucleic acid constitutes at least about 0.5 %, preferably at least about 2%, and more preferably at least about 5% by weight of total nucleic acid present in a given fraction. A partially pure nucleic acid constitutes at least about 10%, preferably at least about 30%, and more preferably at least about 60% by weight of total nucleic acid present in a given fraction. A pure nucleic acid constitutes at least about 80%, preferably at least about 90%, and more preferably at least about 95% by weight of total nucleic acid present in a given fraction.

The invention provides efficient methods of identifying pharmacological agents or drugs which are active at the level of hNFAT modulatable cellular function, particularly hNFAT mediated interleukin signal transduction. Generally, these screening methods involve assaying for compounds which interfere with hNFAT activity such as hNFAT-AP1 binding, hNFAT-DNA binding, etc. The methods are amenable to automated, cost-effective high throughput drug screening and have immediate application in a broad range of domestic and international pharmaceutical and biotechnology drug development programs.

Target therapeutic indications are limited only in that the target cellular function (e.g. gene expression) be subject to modulation, usually inhibition, by disruption of the formation of a complex (e.g. transcription complex) comprising a hNFAT or hNFAT fragment and one or more natural hNFAT intracellular binding targets. Since a wide variety of genes are subject to hNFAT regulated gene transcription, target indications may include infection, metabolic disease, genetic disease, cell growth and regulatory disfunction, such as neoplasia, inflammation, hypersensitivity, etc. Frequently, the target indication is related to either immune dysfunction or selective immune suppression.

A wide variety of assays for binding agents are provided including labelled in vitro protein-protein and protein-DNA binding assay, electrophoretic mobility shift assays, immunoassays for protein binding or transcription complex formation, cell based assays such as one, two and three hybrid screens, expression assays such as transcription assays, etc. For example, three-hybrid screens are used to rapidly examine the effect of transfected nucleic acids, which may, for example, encode combinatorial peptide libraries or antisense molecules, on the intracellular binding of hNFAT or hNFAT fragments to intracellular hNFAT targets. Convenient reagents for such assays (e.g. GAL4 fusion partners) are known in the art.

hNFAT or hNFAT fragments used in the methods are usually added in an isolated, partially pure or pure form and are typically recombinantly produced. The hNFAT or fragment may be part of a fusion product with another peptide or polypeptide, e.g. a polypeptide that is capable of providing or enhancing protein--protein binding, sequence-specific nucleic acid binding or stability under assay conditions (e.g. a tag for detection or anchoring).

The assay mixtures comprise at least a portion of a natural intracellular hNFAT binding target such as AP1 or a nucleic acid comprising a sequence which shares sufficient sequence similarity with a gene or gene regulatory region to which the native hNFAT naturally binds to provide sequence-specific binding of the hNFAT or hNFAT fragment. Such a nucleic acid may further comprise one or more sequences which facilitate the binding of a second transcription factor or fragment thereof which cooperatively binds the nucleic acid with the hNFAT (i.e. at least one increases the affinity or specificity of the DNA binding of the other). While native binding targets may be used, it is frequently preferred to use portions (e.g. peptides, nucleic acid fragments) or analogs (i.e. agents which mimic the hNFAT binding properties of the natural binding target for the purposes of the assay) thereof so long as the portion provides binding affinity and avidity to the hNFAT conveniently measurable in the assay. Binding sequences for other transcription factors may be found in sources such as the Transcription Factor Database of the National Center for Biotechnology Information at the National Library for Medicine, in Faisst and Meyer (1991) Nucleic Acids Research 20, 3-26, and others known to those skilled in this art.

Where used, the nucleic acid portion bound by the peptide(s) may be continuous or segmented and is usually linear and double-stranded DNA, though circular plasmids or other nucleic acids or structural analogs may be substituted so long as hNFAT sequence-specific binding is retained. In some applications, supercoiled DNA provides optimal sequence-specific binding and is preferred. The nucleic acid may be of arty length amenable to the assay conditions and requirements. Typically the nucleic acid is between 8 bp and 5 kb, preferably between about 12 bp and 1 kb, more preferably between about 18 bp and 250 bp, most preferably between about 27 and 50 bp. Additional nucleotides may be used to provide structure which enhances or decreased binding or stability, etc. For example, combinatorial DNA binding can be effected by including two or more DNA binding sites for different or the same transcription factor on the oligonucleotide. This allows for the study of cooperative or synergistic DNA binding of two or more factors. In addition, the nucleic acid can comprise a cassette into which transcription factor binding sites are conveniently spliced for use in the subject assays.

The assay mixture also comprises a candidate pharmacological agent. Generally a plurality of assay mixtures are run in parallel with different agent concentrations to obtain a differential response to the various concentrations. Typically, one of these concentrations serves as a negative control, i.e. at zero concentration or below the limits of assay detection. Candidate agents encompass numerous chemical classes, though typically they are organic compounds; preferably small organic compounds. Small organic compounds have a molecular weight of more than 50 yet less than about 2,500, preferably less than about 1000, more preferably, less than about 500. Candidate agents comprise functional chemical groups necessary for structural interactions with proteins and/or DNA, and typically include at least an amine, carbonyl, hydroxyl or carboxyl group, preferably at least two of the functional chemical groups, more preferably at least three. The candidate agents often comprise cyclical carbon or heterocyclic structures and/or aromatic or polyaromatic structures substituted with one or more of the forementioned functional groups. Candidate agents are also found among biomolecules including peptides, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof, and the like. Where the agent is or is encoded by a transfected nucleic acid, said nucleic acid is typically DNA or RNA.

Candidate agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural and synthetically produced libraries and compounds are readily modified through conventional chemical, physical, and biochemical means. In addition, known pharmacological agents may be subject to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc., to produce structural analogs.

A variety of other reagents may also be included in the mixture. These include reagents like salts, buffers, neutral proteins, e.g. albumin, detergents, etc. which may be used to facilitate optimal protein-protein and/or protein-nucleic acid binding and/or reduce non-specific or background interactions, etc. Also, reagents that otherwise improve the efficiency of the assay, such as protease inhibitors, nuclease inhibitors, antimicrobial agents, etc. may be used.

The resultant mixture is incubated under conditions whereby, but for the presence of the candidate pharmacological agent, the hNFAT specifically binds the cellular binding target, portion or analog. The mixture components can be added in any order that provides for the requisite bindings. Incubations may be performed at any temperature which facilitates optimal binding, typically between 4.degree. and 40.degree. C., more commonly between 15.degree. and 40.degree. C. Incubation periods are likewise selected for optimal binding but also minimized to facilitate rapid, high-throughput screening, and are typically between 0.1 and 10 hours, preferably less than 5 hours, more preferably less than 2 hours.

After incubation, the presence or absence of specific binding between the hNFAT and one or more binding targets is detected by any convenient way. For cell-free binding type assays, a separation step is often used to separate bound from unbound components. The separation step may be accomplished in a variety of ways. Conveniently, at least one of the components is immobilized on a solid substrate which may be any solid from which the unbound components may be conveniently separated. The solid substrate may be made of a wide variety of materials and in a wide variety of shapes, e.g. microtiter plate, microbead, dipstick, resin particle, etc. The substrate is chosen to maximize signal to noise ratios, primarily to minimize background binding, for ease of washing and cost.

Separation may be effected for example, by removing a bead or dipstick from a reservoir, emptying or diluting reservoir such as a microtiter plate well, rinsing a bead (e.g. beads with iron cores may be readily isolated and washed using magnets), particle, chromatographic column or filter with a wash solution or solvent. Typically, the separation step will include an extended rinse or wash or a plurality of rinses or washes. For example, where the solid substrate is a microtiter plate, the wells may be washed several times with a washing solution, which typically includes those components of the incubation mixture that do not participate in specific binding such as salts, buffer, detergent, nonspecific protein, etc. may exploit a polypeptide specific binding reagent such as an antibody or receptor specific to a ligand of the polypeptide.

Detection may be effected in any convenient way. For cell based assays such as one, two, and three hybrid screens, the transcript resulting from hNFAT-target binding usually encodes a directly or indirectly detectable product (e.g. galactosidase activity, luciferase activity, etc.). For cell-free binding assays, one of the components usually comprises or is coupled to a label. A wide variety of labels may be employed--essentially any label that provides for detection of bound protein. The label may provide for direct detection as radioactivity, luminescence, optical or electron density, etc. or indirect detection such as an epitope tag, an enzyme, etc. The label may be appended to the protein e.g. a phosphate group comprising a radioactive isotope of phosphorous, or incorporated into the protein structure, e.g. a methionine residue comprising a radioactive isotope of sulfur.

A variety of methods may be used to detect the label depending on the nature of the label and other assay components. For example, the label may be detected bound to the solid substrate or a portion of the bound complex containing the label may be separated from the solid substrate, and thereafter the label detected. Labels may be directly detected through optical or electron density, radiative emissions, nonradiative energy transfers, etc. or indirectly detected with antibody conjugates, etc. For example, in the case of radioactive labels, emissions may be detected directly, e.g. with particle counters or indirectly, e.g. with scintillation cocktails and counters. The methods are particularly suited to automated high throughput drug screening. Candidate agents shown to inhibit hNFAT-target binding or transcription complex formation provide valuable reagents to the pharmaceutical industries for animal and human trials.

As previously described, the methods are particularly suited to automated high throughput drug screening. In a particular embodiment, the arm retrieves and transfers a microtiter plate to a liquid dispensing station where measured aliquots of each an incubation buffer and a solution comprising one or more candidate agents are deposited into each designated well. The arm then retrieves and transfers to and deposits in designated wells a measured aliquot of a solution comprising a labeled transcription factor protein. After a first incubation period, the liquid dispensing station deposits in each designated well a measured aliquot of a biotinylated nucleic acid solution. The first and/or following second incubation may optionally occur after the arm transfers the plate to a shaker station. After a second incubation period, the arm transfers the microtiter plate to a wash station where the unbound contents of each well is aspirated and then the well repeatedly filled with a wash buffer and aspirated. Where the bound label is radioactive phosphorous, the arm retrieves and transfers the plate to the liquid dispensing station where a measured aliquot of a scintillation cocktail is deposited in each designated well. Thereafter, the amount of label retained in each designated well is quantified.

In more preferred embodiments, the liquid dispensing station and arm are capable of depositing aliquots in at least eight wells simultaneously and the wash station is capable of filling and aspirating ninety-six wells simultaneously. Preferred robots are capable of processing at least 640 and preferably at least about 1,280 candidate agents every 24 hours, e.g. in microliter plates. Of course, useful agents are identified with a range of other assays (e.g. gel shifts, etc.) employing the subject hNFAT and hNFAT fragments.

The subject hNFAT and hNFAT fragments and nucleic acids provide a wide variety of uses in addition to the in vitro binding assays described above. For example, cell-based assays are provided which involve transfecting a T-cell antigen receptor expressing cell with an hNFAT inducible reporter such as luciferase. Agents which modulate hNFAT mediated cell function are then detected through a change in the reporter.

The following examples are offered by way of illustration and not by way of limitation.

EXPERIMENTAL

Investigation of the antigen inducible expression of the IL-2 gene led to the discovery of the regulatory transcription factor NFAT (Nuclear Factor of Activated T cells) (Durand et al. 1988; Shaw et al. 1988). Like several other transcription factors involved in mediating signal transduction, the activity of NFAT is regulated by subcellular localization. In resting T cells NFAT activity is restricted to cytoplasm; stimulation of the T cell receptor leads to translocation of NFAT to the nucleus. Movement of NFAT to the nucleus is dependent on the activation of the calcium-regulated phosphatase calcineurin (Clipstone and Crabtree 1992). The immunosuppressive drugs cyclosporin and FK506 inhibit the activity of calcineurin, and thereby prevent the nuclear localization of NFAT and subsequent activation of cytokine gene expression (reviewed in (Schreiber and Crabtree 1992).

Activation of the T cell antigen receptor induces two signalling pathways required for IL-2 induction, one is the cyclosporin-sensitive, calcium-dependent pathway and the other relies on the activation of protein kinase C (PKC). Antigenic stimulation of these pathways can be mimicked by treating cells with a calcium ionophore and a phorbol ester. The PKC-inducible activity was found to be mediated by fos and jun proteins (Jain et al. 1992; Northrop et al. 1993). The NFAT binding site in the IL-2 promoter is adjacent to a weak binding site for AP-1 proteins, and NFAT and AP-1 proteins bind cooperatively to this composite element (Jain et al. 1993; Northrop et al. 1993). The transcriptional activation mediated by AP-1 proteins through this site appears to be critical for IL-2 expression in activated T cells. There are several different combinations of fos and jun family members that can interact with NFAT to bind DNA (Boise et al. 1993; Northrop et al. 1993; Jain et al. 1994; Yaseen et al. 1994). Therefore, the composition of the AP-1 complex that interacts with NFAT may vary in different cell types and different stages of T cell activation. NFAT was originally reported to be a T cell specific transcription factor critical for the restricted expression of IL-2 (Shaw et al. 1988). More recently, NFAT activity was detected in B cells (Brabletz et al. 1991; Yaseen et al. 1993; Choi et al. 1994; Venkataraman et al. 1994). This is consistent with the finding that, in transgenic mice, the major sites of expression of a reporter gene regulated by the IL-2 NFAT/AP-1 site are activated T and B cells (Verweij et at. 1990).

In addition to IL-2, NFAT sites have been discovered in the promoters of several other cytokine genes, including IL-4 (Chuvpilo et al. 1993; Szabo et al. 1993; Rooney et al. 1994), IL-3 (Cockerill et al. 1993), GM-CSF (Masuda et al. 1993), and TNF-a (Goldfeld et al. 1993). Thus, it appears that NFAT proteins are involved in the coordinate regulation of many different cytokines in activated lymphocytes. As with IL-2, most of the NFAT sites in other cytokine promoters are composite elements that also contain AP-1 binding sites (Rao, 1994).

Distinct genes encoding NFAT proteins have now been isolated (Jain et al. 1993; McCaffrey et al. 1993; Northrop et al. 1994; Hoey et al., in press). Two of these genes, designated NFATp and NFATc, encode related proteins that are highly similar to each other within a 290 amino acid domain. This NFAT homology region shares weak sequence similarity with the DNA binding and dimerization domain of the rel family of transcription factors (reviewed in (Nolan 1994). There is evidence that both NFATp and NFATc may be involved in mediating transcriptional regulation in activated T cells. For example, NFATp forms a specific complex on DNA with fos and jun that activates transcription in vitro (McCaffrey et al. 1993). NFATc has been shown to activate IL-2 expression by a cotransfection assay in T cells (Northrop et al. 1994). Furthermore, both proteins appears to be modified by calcineurin (Jain et al 1993; Northrop et al. 1994). In addition to NFATp and NFATc, we have isolated two new members of the human NFAT gene family. We have used these clones to examine the tissue distribution of the different NFAT genes. We have also expressed and purified the DNA binding domains of the NFAT family proteins and investigated their biochemical activities.

Results

1. Cloning of Human NFAT Genes

cDNA libraries were prepared from Jurkat T cells and human peripheral blood lymphocytes, and screened using a probe derived from the rel similarity region of the murine NFATp gene (McCaffrey et al. 1993). Cross-hybridizing clones were isolated, sequenced, and determined to be derived from 4 distinct genes.

One of the genes isolated in this study is related to the murine NFATp gene (McCaffrey et al. 1993), and another is identical to the NFATc gene (Northrop et al. 1994). We have isolated two classes of NFATp cDNAs which are the result of alternative splicing upstream of the rel domain. One form is similar to the cDNA reported by McCaffrey et al., while the other is alternatively spliced downstream of the rel similarity region; in particular, this form is missing an exon encoding the region near the N-terminus of the protein (SEQ ID NO: 1, base pairs 357-867) and has a different initiating methionine (SEQ ID NO: 1, base pairs 880-882).

In addition to these previously identified genes, we cloned two novel members of the NFAT gene family, hereby designated as NFAT3 and NFAT4. The NFAT3 sequence was obtained from three overlapping cDNAs spanning 2880 bp, and deduced to encode a protein of 902 amino acids. We obtained three classes of NFAT4 cDNAs that resulted from alternative splicing downstream of the rel homology domain. These three types of cDNAs encode proteins that vary in sequence and length at their C-terminal ends. The three forms are designated NFAT4a, NFAT4b, and NFAT4c. The positions of splice junctions in the coding regions are after proline 699 in NFAT4a and after valine 700 and proline 716 in NFAT4b and NFAT4c.

All of the NFAT genes are at least 65% identical to each other within a 290 amino acid domain. This domain is related to the DNA binding and dimerization domain of the rel family of transcription factors (Nolan 1994; Northrop et al. 1994). Among the different NFAT genes, the N-terminal and central portions of the rel similarity domain are more highly conserved than the C-terminus.

Aside from the strikingly similar rel domains shared by all four NFAT genes, the NFAT family members have smaller regions of sequence similarity on the amino terminal side of the rel domains. The amino terminal regions of NFAT4 and NFATc have several regions of significant similarity. The two largest regions contain 23 of 41 and 24 of 45 identical amino acids between the two proteins. Both of these regions are rich in serine and proline residues. NFATp and NFAT3 also have some similarity to the other NFAT proteins in this region, although it is less extensive than that shared between NFAT4 and NFATc. The homology between NFAT3 and NFAT4 extends about 25 amino acids upstream of the rel similarity region.

2. Expression Patterns of the NFAT Genes

On tile basis of previous reports, expression of NFAT genes was expected to be restricted to lymphocytes (Shaw et al. 1988; Verweij et al. 1990; McCaffrey et al. 1993; Northrop et al. 1994). The expression of each NFAT gene was tested by Northern blot using RNA from sixteen different human tissues. For NFATp, expression of an mRNA approximately 7.5 kb was detected in almost all human tissues. The expression was slightly higher in PBLs and placenta. NFATc expression was also detected at a low level in several different tissues. The NFATc probe hybridized to two bands of approximately 2.7 and 4.5 kb. Surprisingly, the 4.5 kb NFATc transcript was strongly expressed in skeletal muscle. The 2.7 kb mRNA appears to correspond to the previously described NFATc clone (Northrop et al. 1994).

NFAT3 exhibited a very complicated expression pattern with at least 3 major RNA bands between 3 and 5 kb. The major sites of NFAT3 expression were observed outside the immune system. NFAT3 was highly expressed in placenta, lung, kidney, testis and ovary. In contrast, NFAT3 expression was very weak in spleen and thymus and undetectable in PBLs.

NFAT4 was expressed predominately as a 6.5 kb message. Like NFATc it was strongly expressed in skeletal muscle. NFAT4 also displayed relatively high expression in thymus. The probe for the NFAT4 northerns contained the 3' half of the NFAT homology region as well as downstream regions from the NFAT4c class of cDNA. This probe should hybridize to all three classes of NFAT4 transcripts. Only one form is detected in the Northern blots, suggesting that the 4c class is the most abundant transcript.

These results indicate that each of the NFAT genes is expressed in a distinct tissue-specific pattern. Furthermore, none of the NFAT genes are restricted to lymphocytes.

3. DNA Binding Activity of the NFAT Proteins

The rel similarity regions along with a small amount of flanking sequences of each of the four classes of NFAT proteins were expressed in E. coli. Each of the 4 proteins was well expressed and soluble. The proteins were purified to near homogeneity by DNA affinity chromatography (Kadonaga and Tjian 1986). The binding site used for purification was a high affinity NFAT site derived from the IL-4 promoter with the core binding sequence GGAAAATTTT (SEQ ID NO: 15) (Rooney et al. 1994).

The binding specificities of the NFAT proteins were tested on two known functional binding sites, the IL-4 promoter NFAT site and the NFAT binding site in the distal antigen response element from the IL-2 promoter (Durand et al. 1988; Shaw et al. 1988). All the proteins were able to bind the IL-4 promoter site. NFATp, NFATc, and NFAT3 recognized this sequence with very similar affinity, while NFAT4 bound this sequence with lower affinity (>10-fold) than the other three proteins in this assay. NFAT4 protein may have a different optimum binding sequence than the other NFAT proteins.

The same amounts of the four NFAT proteins were tested on the NFAT binding site from the IL-2 promoter. This NFAT site (GGAAAAACTG) (SEQ ID NO:16) has three differences relative to the IL-4 site which make it a weaker site for all four NFAT proteins. The NFAT proteins differ in their ability to recognize this site independently. NFATp had the highest relative affinity for the IL-2 binding site, while NFATc and NFAT3 bound weakly to this site and NFAT4 binding was not detectable in this assay.

The IL-2 NFAT site is part of a composite element that is adjacent to a weak AP-1 site (TGTTTCA) (Jain et al. 1992; Northrop et al. 1993). To determine if there were any differences in the ability of NFAT proteins to interact with AP-1, the four NFAT proteins were tested with AP-1 for binding to the IL-2 site. When tested alone all the NFAT proteins, as well as the AP-1 proteins, bound relatively weakly to the IL-2 composite element. The combination of c-jun and fra1 with each of the four NFAT proteins resulted in highly cooperative DNA binding. In the presence of the AP-1 protein the four NFAT proteins bound to the IL-2 site with very similar affinity. In all cases, jun homodimers were not as effective as jun-fra1 heterodimers in promoting cooperative binding in the gel shift assay. These results indicate that the DNA binding and protein interaction specificity of the NFAT proteins are very similar. Indeed, the interactions of the four NFAT proteins with these AP-1 proteins appear to be identical. NFAT4 did not bind independently to this site, but recognized this site with the same affinity as the other NFAT proteins in the presence of AP-1.

4. Transcriptional Activation by the NFAT Poteins

Having established that the DNA binding properties of the four NFAT proteins are quite similar, we investigated their transcriptional activation potentials. We used a transient transfection assay into Jurkat T cells to measure the ability of the NFAT proteins to activate the IL-2 promoter. The IL-2 promoter was chosen because it is a critical regulatory target for NFAT and has at least two functional NFAT binding sites (Randak et al. 1990). Activation of this promoter by antigenic stimulation can be mimicked by treatment with phorbol esters, such as phorbol 12-myristate 13 acetate (PMA), together with ionomycin, a calcium ionophore.

Each of the four NFAT genes was transfected into Jurkat cells, and their ability to activate the IL-2 promoter was tested with various combinations of PMA and ionomycin. Treatment of the cells with PMA plus ionomycin induced strong activation by the endogenous NFAT proteins in Jurkat cells. Transfection of each of the four of the NFAT genes resulted in an additional stimulation the IL-2 promoter between 4- and 8-fold. Activation of the IL-2 promoter by each of the NFAT proteins was dependent on both PMA and ionomycin.

We also tested the ability of NFAT to activate transcription in COS and HepG2 cells using a synthetic reporter gene consisting three copies of an NFAT/AP-1 composite element. Transfection of each of the four NFAT into HepG2 cells resulted in activation of the reporter gene of at least 20-fold in the presence of PMA and ionomycin. In contrast to Jurkat cells, NFAT3 was more potent than the others in the HepG2 transfections, resulting in 140-fold activation. Another difference between the results of HepG2 and Jurkat cells is that the NFAT proteins appeared to activate transcription in the absence of PMA or calcium ionophore.

In COS cells NFAT3 produced a striking 50-fold activation that was observed independently of PMA and ionomycin treatment. NFAT3 was found to stimulate transcription in COS cells much more strongly than the other proteins.

5. NFAT Proteins are Active as Monomers

There are many similar features of the NFAT and rel families of transcription factors. Rel proteins form homo- and heterodimers in solution, and dimerization is required for DNA binding (reviewed in Baeuerle and Henkel 1994). The C-terminal half of the rel homology domain is thought to be involved in mediating dimerization. Since the similarity between NFAT and the rel families extends throughout the 300 amino acid rel domain, and the rel domain of the NF-kB proteins is sufficient for dimer formation, we expected that the NFAT proteins might also be function as dimers. To test this idea we determined the native masses of the NFAT proteins by gel filtration chromatography and glycerol gradient centrifugation. For these experiments we used the rel similarity regions of NFATp and NFATc that were expressed in E. coli and purified by DNA affinity chromatography. The molecular weights of these proteins are 40.4 and 35.6 kD, respectively. As a control we used purified NF-kB p50 protein that is known to exist as a stable dimer in solution (Baeuerle and Baltimore 1989). The p50 protein is 45.8 kD calculated from its amino acid sequence.

On both the gel filtration column and the glycerol gradient, the NFATp and NFATc rel domains migrated at a position close to their actual molecular weight. Under the same conditions, p50 behaved as species that was larger than its monomer molecular weight. The data from the gel filtration column was used to calculate the Stokes radius of each protein, and the S values were determined by glycerol gradient sedimentation. These two properties were used to calculate the apparent molecular size of the proteins (Siegel and Monty 1966; Thompson et al. 1991). The apparent molecular sizes of the NFATp and NFATc rel domains were determined to be 42 kD and 32 kD respectively. These values are close to the monomer molecular weight for both NFAT proteins. As expected, p50 exhibited an apparent molecular size close to that of a dimer.

After determining that NFAT rel domains were monomers in solution, we then considered the possibility that NFAT proteins might form dimers when bound to DNA. To address this question we carried out gel mobility shift assays with two different sized versions of NFATc translated in vitro (Hope and Struhl 1987). The shorter version contains the rel similarity region and a small amount of flanking residues and is referred to as NFATc-309. This construct is equivalent to the one that was expressed in E. coli. The larger version, NFATc-589, contains additional N-terminal sequences. When expressed individually in a rabbit reticulocyte lysate both versions of NFATc were active and produced protein-DNA complexes with different mobilities. When the two different NFATc proteins were mixed by co-translation the same protein-DNA complexes were apparent and no intermediate species was detectable, as would be expected if the proteins were forming dimers on the DNA. These results suggest that NFAT proteins are capable of sequence-specific DNA binding as monomers.

Methods

1. Isolation of Human NFAT Clones

Peripheral blood lymphocytes (PBLs) were isolated from 2 units of blood (obtained from Irwin Memorial Blood Bank, San Francisco) by fractionation on sodium metrizoate/polysaccharide (Lymphoprep, Nycomed) gradients. Jurkat T cells were grown in RPMI+10% fetal bovine serum. Total RNA was isolated from Jurkat cells or peripheral blood lymphocytes according to the Guanidinium-HCl method (Chomczynski and Sacchi 1987). Poly-A+RNA was purified using oligo-dT magnetic beads (Promega). Random primed and oligo-dT primed libraries were prepared from both Jurkat and PBL RNA samples. The cDNA libraries were constructed in the vector Lambda ZAPII (Stratagene) according to the protocol supplied by the manufacturer. The cDNA was size selected for greater than 1 kb by electrophoresis a on 5 % polyacrylamide gel prior to ligation. Each library contained approximately 2.times.10.sup.6 recombinant clones. Each of the four libraries was screened independently under the same conditions.

The probe for the initial library screen was a 372 bp fragment derived by PCR from the C-terminal half of the rel homology domain of the mouse NFATp gene. This region corresponds to amino acids 370 through 496 in the published mNFATp sequence (McCaffrey et al. 1993). The fragment was labeled by random priming and hybridized in 1M NaCl, 50 mM Tris pH 7.4, 2 mM EDTA, 10.times. Denhardt's, 0.05% SDS, and 50 mg/ml salmon sperm DNA at 60.degree. C. The filters were washed first in 2.times. SSC, 0.1% SDS, and then in 1.times. SSC, 0.1% SDS at 60.degree. C. Hybridizing clones were purified and converted into Bluescript plasmid DNA clones. The DNA sequence was determined using thermal cycle sequencing and the Applied Biosystems 373A sequencer. Approximately 50 clones were isolated from the first set of screens. Sequence analysis and cross-hybridization experiments indicated that these clones were derived from 4 distinct genes. For NFAT4, additional cDNA clones were obtained from a skeletal muscle cDNA library (Stratagene). The 5' ends of the cDNA clones were obtained from a Jurkat cDNA library prepared as described above with gene specific primers for each of the NFAT genes.

2. Northerns

The northern blots with mRNA isolated from human tissues were purchased from Clontech. DNA probes were labeled by random priming and hybridized in 5.times. SSPE, 10.times. Denhardt's, 50% formamide, 2% SDS, 100 mg/ml salmon sperm DNA at 42.degree. C. The filters were washed in 2.times. SSC/0.05% SDS at room temperature, and subsequently in 0.1.times. SSC/0.1% SDS at 60.degree. C. For NFATp the probe was 1.2 kb cDNA fragment containing the entire rel similarity region of NFATp. For NFATc, the probe was a 291 nucleotide PCR fragment corresponding to the 3' end of rel similarity region (amino acids 597 to 693 (Northrop et al. 1994). For NFATc, a different set of blots was hybridized with a 0.8 kb cDNA fragment located upstream of the rel domain. The two different NFATc probes produced identical results. For NFAT3, the probe was a 0.6 kb fragment located downstream of the rel similarity region corresponding to the region encoding amino acid 720 through the 3' end of the clone. For NFAT4, the probe was a 1.3 kb cDNA fragment corresponding to residue 549 to 963 from the 4c class of cDNAs.

3. Protein Expression and Purification

E. coli expression vectors for each NFAT protein were constructed in the T7 polymerase expression vector pT7-HMK, which has an eight amino acid heart muscle kinase (hmk) site at the N-terminus. NdeI sites were introduced by PCR using mutagenic oligonucleotides in the coding regions upstream of the NFAT rel domains, and these restriction sites were subsequently used for cloning into pT7-HMK. The sizes of the different proteins (without the hmk sequences) are as follows: NFATp, 353 amino acids (the residues homologous to 185 through 537 according to McCaffrey et al. 1993); NFATc, 309 amino acids (amino acids 408 through 716 according to Northrop et al. 1994); NFAT3, 345 amino acids (residues 400 through 744); NFAT4, 316 amino acids (residues 393 through 708).

Proteins were expressed using the T7 polymerase expression system in the strain BL21(DE3) (Studier and Moffat 1986). Expression was induced by addition of 0.4 mM IPTG, and the cultures were shaken for 4 hours at room temperature. The cells were harvested, washed in PBS, resuspended in 0.4M KCl-HEG (25 mM HEPES pH 7.9; 0.1 mM EDTA; 10% glycerol; 0.2% NP-40; 2 mM DTT, 0.2 mM PMSF, 0.2 mM sodium metabisulfite) and lysed by two cycles of freeze- thawing followed by sonication. The lysate was spun in 10 min to remove insoluble material. NFAT proteins were purified from the soluble fractions of the extracts by DNA affinity chromatography (Kadonaga and Tjian 1986). The binding site sequence for the affinity resin was from the IL-4 promoter, TACATTGGAAAATTTTATTACAC (SEQ ID NO:17). The DNA was biotinylated on one strand and coupled to avidin agarose beads (Sigma) at a concentration of approximately 1 mg DNA/ml. Approximately 10 mg of E. coli extracts containing the recombinant NFAT proteins were loaded on 1.5 ml DNA columns equilibrated with 0.1M KCl-HEG. The columns were washed successively with 0.1, 0.2, and 0.4M HEG. The specifically bound NFAT proteins were eluted with 1.0M KCl-HEG.

Fra-1 was expressed in E. coli from the vector pET11 (Novagen). The protein was purified from the soluble fraction to approximately 80% homogeneity by fractionation on heparin-sepharose. c-Jun protein was expressed in E. coli and purified from the insoluble portion of the extract as previously described (Bohmann and Tjian, 1989). The concentrations of the purified proteins were determined by comparing the intensity of coomassie staining with the staining intensity of BSA standards.

4. DNA Binding Experiments

Electrophoretic mobility shift assays were performed with the indicated amounts of proteins in 50 mM KCl, 25 mM HEPES, 0.05 mM EDTA, 5% glycerol, 1 mM DTT with 1 mg of poly(dI-dC) and 100 ng of BSA. The binding reactions and electrophoresis were carried out at room temperature. The samples were run on a 5% polyacrylamide, 0.5.times. TBE gel at 200 V.

5. Transfections

The full-length coding regions for each of the NFAT genes were subcloned into the RSV expression vector pREP4 (Invitrogen). The reporter plasmid was pXIL2-Luc (constructed by Jim Fraser). It contains the IL-2 promoter (-326 to +47, as in Durand et al 1988) upstream of the luciferase gene. Approximately 1.times.10.sup.6 Jurkat cells were transiently transfected by lipofection (Lipofectin, Gibco/BRL). Twenty hours after transfection the cells were treated with 25 ng/ml PMA and 2 mM ionomycin, and the cells were harvested 8 hours after induction. Transfection efficiencies were standardized by co-transfection of pRSV-bgal and subsequent determination of bgal activity. Each transfection contained 2 mg of expression vector, 5 mg of luciferase reporter, and 1 mg of bgal plasmid and 10 ml of lipofectin. COS-7 and HepG2 cells were transfected by a modification of the calcium phosphate method (Chen and Okayama 1987). The reporter gene contained three copies of the antigen response element (-286 to -257) upstream of the herpes virus tk minimal promoter (-50 to +28) in the luciferase vector pGL2 (Promega).

6. Gel Filtration Columns and Glycerol Gradients

Protein samples were run on a 2.4 ml Superdex-200 column using the Pharmacia Smart system. The column was equilibrated with 0.5M KCl-HEG at a flow rate of 80 ml/min. The elution volumes of purified NFATc, NFATp, and p50 were determined relative to those of molecular weight standards. Purified p50 was provided by Zhaodan Cao. The following molecular weight standards (10 mg) were chromatographed on separate runs: thyroglobulin (669 kD), b-amylase (200 kD), BSA (66 kD), carbonic anhydrase (29 kD), and cytochrome c (12 kD). The elution volume (V.sub.e) was converted to K.sub.av by the equation, K.sub.av =(V.sub.e -V.sub.o) /V.sub.i, where V.sub.o is the void volume and V.sub.i is the included volume. The Stokes radii were determined from a plot of (-log K.sub.av).sup.1/2 vs. the Stokes radii of the standards (Ackers 1970).

The S values were determined by glycerol gradient centrifugation. Five ml 10-30% glycerol gradients were prepared using a Beckman density gradient former. The samples were centrifuged in a SW50Ti rotor at 39,000 rpm for 40 hours. After centrifugation, 200-ml fractions were collected and analyzed by gel electrophoresis and coomassie staining. The S values were determined by their sedimentation positions relative to the standards. Native molecular sizes were determined from the Stokes radii (a), S values (s), and the partial specific volumes (V) by the method of Siegel and Monty using the equation M=6pNas/1-V (Siegel and Monty 1966, Thompson et al. 1991).

7. References Cited in Experimental Section

Ackers (1970) Adv. Prof. Chem. 24:343-446; Baeuerle and Baltimore (1989) Genes & Dev. 3:1689-1698; Baeuerle and Henkel (1994) Annu. Rev. Immunol. 12:141-179; Boise et al. (1993 ) Mol. Cell. Biol. 13:1911-1919; Brabletz et al. (1991) Nucl. Acids Res. 19:61-67; Chen and Okayama (1987) Mol. Cell. Biol. 7:2745-2752; Choi et al. (1994) Immunity 1:179-187; Chomczynski and Sacchi (1987) Anal. Biochem. 162: 156-159; Chuvpilo et al. (1993) Nucl. Acids Res. 21:5694-5704; Clipstone and Crabtree (1992) Nature 357:695-697; Cockerill et al. (1993) Proc. Natl. Acad. Sci. USA 90:2466-2470; Durand et al. (1988) Mol. Cell. Biol. 8:1715-1724; Goldfeld et al. (1993) J. Exp. Med. 178:1365-1379; Grabstein et al. (1994) Science 264:965-968; Hohlfeld and Engel (1994) Immunol. Today 15:269-274; Hoyos et al. (1989) Science 244:457-460; Hope and Struhl (1987) EMBO J. 6:2781-2784; Jain et al. (1992) Nature 356:801-803; Jain et al. (1993) Nature 365:352-355; Jain et al. (1993) J. Immunol. 151:837-848; Jain et al. (1994) Mol. Cell. Biol. 14:1566-1574; Kadonaga and Tjian (1986) Proc. Natl. Acad. Sci. USA 83:5889-5893; Masuda (1993) Mol. Cell. Biol. 13:7399-7407; McCaffrey et al. (1993) Science 262:750-754; McCaffrey et al. (1993) J. Biol. Chem. 268:3747-3752; Mouzaki and Rungger (1994) Blood 84:2612-2621; Nolan (1994) Cell 77:795-798; Northrop (1994) Nature 369:497-502; Northrop (1993) J. Biol. Chem. 268:2917-2293; Randak (1990) EMBO J. 9:2529-2536; Rooney (1994) EMBO J. 13:625-633; Schreiber and Crabtree (1992) Immunol. Today 13:136-142; Shaw (1988) Science 241:202-205; Siegel and Monty (1966) Biochim. Biophys. Acta 112:346-362; Studier and Moffat (1986) J. Mol. Biol. 189:113-130; Szabo (1993) Mol. Cell. Biol. 13:4793-4805; Thompson et al . (1991) Science 253:762-768; Venkataraman et al. (1994) Immunity 1:189-196; Verweij et al. (1990) J. Biol. Chem 265:15788-15795; Yaseen et al. (1994) Mol. Cell. Biol. 14:6886-6895; and Yaseen et al. (1993) J. Biol. Chem. 268:14285-14293.

The following examples are offered by way of illustration and not by way of limitation.

EXAMPLES

1. Protocol for hNFAT-hNFAT Dependent Transcription Factor Binding Assay.

A. Reagents:

hNFAT: 20 .mu.g/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hr, RT.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktail of protease inhibitors.

.sup.33 P hNFAT 10.times. stock: 10.sup.-8 -10.sup.-6 M "cold" hNFAT homolog supplemented with 200,000-250,000 cpm of labeled hNFAT homolog (Beckman counter). Place in the 4.degree. C. microfridge during screening.

Protease inhibitor cocktail (1000.times.): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVo.sub.3 (Sigma #S-6508) in 10 ml of PBS.

B. Preparation of Assay Plates:

Coat with 120 .mu.l of stock NF-AT per well overnight at 4.degree. C.

Wash 2.times. with 200 .mu.l PBS.

Block with 150 .mu.l of blocking buffer.

Wash 2.times. with 200 .mu.l PBS.

C. Assay:

Add 80 .mu.l assay buffer/well.

Add 10 .mu.l compound or extract.

Add 10 .mu.l .sup.33 P-NFAT (20,000-25,000 cpm/0.3 pmoles/well=3.times.10.sup.-9 M final concentration).

Shake at 25.degree. C. for 15 min.

Incubate additional 45 min. at 25.degree. C.

Stop the reaction by washing 4.times. with 200 .mu.l PBS.

Add 150 .mu.l scintillation cocktail.

Count in Topcount.

D. Controls for all Assays (Located on each Plate):

a. Non-specific binding (no hNFAT added)

b. cold hNFAT at 80% inhibition.

2. Protocol for hNFAT-AP1 Dependent Transcription Factor Binding Assay.

A. Reagents:

fos-jun heterodimers (junB and fra1): 20 .mu.g/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hr, RT.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktail of protease inhibitors.

.sup.33 P hNFAT 10.times. stock: 10.sup.-8 -10.sup.6 M "cold" hNFAT homolog supplemented with 200,000-250,000 cpm of labeled hNFAT homolog (Beckman counter). Place in the 4.degree. C. microfridge during screening.

Protease inhibitor cocktail (1000.times.): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma #B-6056), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVo.sub.3 (Sigma #S-6508) in 10 ml of PBS.

B. Preparation of Assay Plates:

Coat with 120 .mu.l of stock fos-jun heterodimers per well overnight at 4.degree. C.

Wash 2.times. with 200 .mu.l PBS.

Block with 150 .mu.l of blocking buffer.

Wash 2.times. with 200 .mu.l PBS.

C. Assay:

Add 80 .mu.l assay buffer/well.

Add 10 .mu.l compound or extract.

Add 10 .mu.l .sup.33 P-NFAT (20,000-25,000 cpm/0.3 pmoles/well=3.times.10.sup.-9 M final concentration).

Shake at 25.degree. C. for 15 min.

Incubate additional 45 min. at 25.degree. C.

Stop the reaction by washing 4.times. with 200 .mu.l PBS.

Add 150 .mu.l scintillation cocktail.

Count in Topcount.

D. Controls for all assays (located on each plate):

a. Non-specific binding (no hNFAT added)

b. cold hNFAT at 80% inhibition.

3.Protocol for hNFAT-fos-jun Dependent Transcription Factor--DNA Binding Assay.

A. Reagents:

Neutralite Avidin: 20 .mu.g/ml in PBS.

Blocking buffer: 5% BSA, 0.5% Tween 20 in PBS; 1 hr, RT.

Assay Buffer: 100 mM KCl, 20 mM HEPES pH 7.6, 0.25 mM EDTA, 1% glycerol, 0.5% NP-40, 50 mM BME, 1 mg/ml BSA, cocktail of protease inhibitors.

.sup.33 P hNFAT 10.times. stock: 10.sup.-6 -10.sup.-8 M "cold" hNFAT homolog supplemented with 200,000-250,000 cpm of labeled hNFAT homolog (Beckman counter) and 10.sup.-6 -10.sup.-8 M fos-jun heterodimers. Place in the 4.degree. C. microfridge during screening.

Protease inhibitor cocktail (1000.times.): 10 mg Trypsin Inhibitor (BMB #109894), 10 mg Aprotinin (BMB #236624), 25 mg Benzamidine (Sigma #B-6506), 25 mg Leupeptin (BMB #1017128), 10 mg APMSF (BMB #917575), and 2 mM NaVo.sub.3 (Sigma #S-6508) in 10 ml of PBS.

Oligonucleotide stock: (specific biotinylated). Biotinylated oligo at 17 pmole/.mu.l AP1-NFAT site: (BIOTIN)-GG AGG AAA AAC TGT TTC ATA CAG AAG GCG T (SEQ ID NO:18)

B. Preparation of Assay Plates:

Coat with 120 .mu.l of stock N-Avidin per well overnight at 4.degree. C.

Wash 2.times. with 200 .mu.l PBS.

Block with 150 .mu.l of blocking buffer.

Wash 2.times. with 200 .mu.l PBS.

C. Assay:

Add 40 .mu.l assay buffer/well.

Add 10 .mu.l compound or extract.

Add 10 .mu.l .sup.33 P-NFAT (20,000-25,000 cpm/0.1-10 pmoles/well=10.sup.-9 -10.sup.-7 M final concentration).

Shake at 25.degree. C. for 15 min.

Incubate additional 45 min. at 25.degree. C.

Add 40 .mu.l oligo mixture (1.0 pmoles/40 .mu.l in assay buffer with 1 ng of ss-DNA)

Incubate 1 hr at RT.

Stop the reaction by washing 4.times. with 200 .mu.l PBS.

Add 150 .mu.l scintillation cocktail.

Count in Topcount.

D. Controls for all Assays (Located on each Plate):

a. Non-specific binding (no oligo added)

b. Specific soluble oligo at 80% inhibition.

All publications and patent applications cited in this specification are herein incorporated by reference as if each individual publication or patent application were specifically and individually indicated to be incorporated by reference. Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims. 

    __________________________________________________________________________
    SEQUENCE LISTING
    (1) GENERAL INFORMATION:
    (iii) NUMBER OF SEQUENCES: 18
    (2) INFORMATION FOR SEQ ID NO:1:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 3478 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: CDS
    (B) LOCATION: 223..2987
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1:
    GGAGCAGGAAGCTCGCGCCGCCGTCGCCGCCGCCGCTCAGCTTCCCCGGGCGCGTCCAGG60
    ACCCGCTGCGCCAGGCGCGCCGTCCCCGGACCCGGCGTGCGTCCCTACGAGGAAAGGGAC120
    CCCGCCGCTCGAGCCGCCTCCGCCAGCCCCACTGCGAGGGGTCCCAGAGCCAGCCGCGCC180
    CGCCCTCGCCCCCGGCCCCGCAGCCTTCCCGCCCTGCGCGCCATGAACGCCCCC234
    MetAsnAlaPro
    GAGCGGCAGCCCCAACCCGACGGCGGGGACGCCCCAGGCCACGAGCCT282
    GluArgGlnProGlnProAspGlyGlyAspAlaProGlyHisGluPro
    5101520
    GGGGGCAGCCCCCAAGACGAGCTTGACTTCTCCATCCTCTTCGACTAT330
    GlyGlySerProGlnAspGluLeuAspPheSerIleLeuPheAspTyr
    253035
    GAGTATTTGAATCCGAACGAAGAAGAGCCGAATGCACATAAGGTCGCC378
    GluTyrLeuAsnProAsnGluGluGluProAsnAlaHisLysValAla
    404550
    AGCCCACCCTCCGGACCCGCATACCCCGATGATGTCCTGGACTATGGC426
    SerProProSerGlyProAlaTyrProAspAspValLeuAspTyrGly
    556065
    CTCAAGCCATACAGCCCCCTTGCTAGTCTCTCTGGCGAGCCCCCCGGC474
    LeuLysProTyrSerProLeuAlaSerLeuSerGlyGluProProGly
    707580
    CGATTCGGAGAGCCGGATAGGGTAGGGCCGCAGAAGTTTCTGAGCGCG522
    ArgPheGlyGluProAspArgValGlyProGlnLysPheLeuSerAla
    859095100
    GCCAAGCCAGCAGGGGCCTCGGGCCTGAGCCCTCGGATCGAGATCACT570
    AlaLysProAlaGlyAlaSerGlyLeuSerProArgIleGluIleThr
    105110115
    CCGTCCCACGAACTGATCCAGGCAGTGGGGCCCCTCCGCATGAGAGAC618
    ProSerHisGluLeuIleGlnAlaValGlyProLeuArgMetArgAsp
    120125130
    GCGGGCCTCCTGGTGGAGCAGCCGCCCCTGGCCGGGGTGGCCGCCAGC666
    AlaGlyLeuLeuValGluGlnProProLeuAlaGlyValAlaAlaSer
    135140145
    CCGAGGTTCACCCTGCCCGTGCCCGGCTTCGAGGGCTACCGCGAGCCG714
    ProArgPheThrLeuProValProGlyPheGluGlyTyrArgGluPro
    150155160
    CTTTGCTTGAGCCCCGCTAGCAGCGGCTCCTCTGCCAGCTTCATTTCT762
    LeuCysLeuSerProAlaSerSerGlySerSerAlaSerPheIleSer
    165170175180
    GACACCTTCTCCCCCTACACCTCGCCCTGCGTCTCGCCCAATAACGGC810
    AspThrPheSerProTyrThrSerProCysValSerProAsnAsnGly
    185190195
    GGGCCCGACGACCTGTGTCCGCAGTTTCAAAACATCCCTGCTCATTAT858
    GlyProAspAspLeuCysProGlnPheGlnAsnIleProAlaHisTyr
    200205210
    TCCCCCAGAACCTCGCCAATAATGTCACCTCGAACCAGCCTCGCCGAG906
    SerProArgThrSerProIleMetSerProArgThrSerLeuAlaGlu
    215220225
    GACAGCTGCCTGGGCCGCCACTCGCCCGTGCCCCGTCCGGCCTCCCGC954
    AspSerCysLeuGlyArgHisSerProValProArgProAlaSerArg
    230235240
    TCCTCATCGCCTGGTGCCAAGCGGAGGCATTCGTGCGCCGAGGCCTTG1002
    SerSerSerProGlyAlaLysArgArgHisSerCysAlaGluAlaLeu
    245250255260
    GTTGCCCTGCCGCCCGGAGCCTCACCCCAGCGCTCCCGGAGCCCCTCG1050
    ValAlaLeuProProGlyAlaSerProGlnArgSerArgSerProSer
    265270275
    CCGCAGCCCTCATCTCACGTGGCACCCCAGGACCACGGCTCCCCGGCT1098
    ProGlnProSerSerHisValAlaProGlnAspHisGlySerProAla
    280285290
    GGGTACCCCCCTGTGGCTGGCTCTGCCGTGATCATGGATGCCCTGAAC1146
    GlyTyrProProValAlaGlySerAlaValIleMetAspAlaLeuAsn
    295300305
    AGCCTCGCCACGGACTCGCCTTGTGGGATCCCCCCCAAGATGTGGAAG1194
    SerLeuAlaThrAspSerProCysGlyIleProProLysMetTrpLys
    310315320
    ACCAGCCCTGACCCCTCGCCGGTGTCTGCCGCCCCATCCAAGGCCGGC1242
    ThrSerProAspProSerProValSerAlaAlaProSerLysAlaGly
    325330335340
    CTGCCTCGCCACATCTACCCGGCCGTGGAGTTCCTGGGGCCCTGCGAG1290
    LeuProArgHisIleTyrProAlaValGluPheLeuGlyProCysGlu
    345350355
    CAGGGCGAGAGGAGAAACTCGGCTCCAGAATCCATCCTGCTGGTTCCG1338
    GlnGlyGluArgArgAsnSerAlaProGluSerIleLeuLeuValPro
    360365370
    CCCACTTGGCCCAAGCCGCTGGTGCCTGCCATTCCCATCTGCAGCATC1386
    ProThrTrpProLysProLeuValProAlaIleProIleCysSerIle
    375380385
    CCAGTGACTGCATCCCTCCCTCCACTTGAGTGGCCGCTGTCCAGTCAG1434
    ProValThrAlaSerLeuProProLeuGluTrpProLeuSerSerGln
    390395400
    TCAGGCTCTTACGAGCTGCGGATCGAGGTGCAGCCCAAGCCACATCAC1482
    SerGlySerTyrGluLeuArgIleGluValGlnProLysProHisHis
    405410415420
    CGGGCCCACTATGAGACAGAAGGCAGCCGAGGGGCTGTCAAAGCTCCA1530
    ArgAlaHisTyrGluThrGluGlySerArgGlyAlaValLysAlaPro
    425430435
    ACTGGAGGCCACCCTGTGGTTCAGCTCCATGGCTACATGGAAAACAAG1578
    ThrGlyGlyHisProValValGlnLeuHisGlyTyrMetGluAsnLys
    440445450
    CCTCTGGGACTTCAGATCTTCATTGGGACAGCTGATGAGCGGATCCTT1626
    ProLeuGlyLeuGlnIlePheIleGlyThrAlaAspGluArgIleLeu
    455460465
    AAGCCGCACGCCTTCTACCAGGTGCACCGAATCACGGGGAAAACTGTC1674
    LysProHisAlaPheTyrGlnValHisArgIleThrGlyLysThrVal
    470475480
    ACCACCACCAGCTATGAGAAGATAGTGGGCAACACCAAAGTCCTGGAG1722
    ThrThrThrSerTyrGluLysIleValGlyAsnThrLysValLeuGlu
    485490495500
    ATACCCTTGGAGCCCAAAAACAACATGAGGGCAACCATCGACTGTGCG1770
    IleProLeuGluProLysAsnAsnMetArgAlaThrIleAspCysAla
    505510515
    GGGATCTTGAAGCTTAGAAACGCCGACATTGAGCTGCGGAAAGGCGAG1818
    GlyIleLeuLysLeuArgAsnAlaAspIleGluLeuArgLysGlyGlu
    520525530
    ACGGACATTGGAAGAAAGAACACGCGGGTGAGACTGGTTTTCCGAGTT1866
    ThrAspIleGlyArgLysAsnThrArgValArgLeuValPheArgVal
    535540545
    CACATCCCAGAGTCCAGTGGCAGAATCGTCTCTTTACAGACTGCATCT1914
    HisIleProGluSerSerGlyArgIleValSerLeuGlnThrAlaSer
    550555560
    AACCCCATCGAGTGCTCCCAGCGATCTGCTCACGAGCTGCCCATGGTT1962
    AsnProIleGluCysSerGlnArgSerAlaHisGluLeuProMetVal
    565570575580
    GAAAGACAAGACACAGACAGCTGCCTGGTCTATGGCGGCCAGCAAATG2010
    GluArgGlnAspThrAspSerCysLeuValTyrGlyGlyGlnGlnMet
    585590595
    ATCCTCACGGGGCAGAACTTTACATCCGAGTCCAAAGTTGTGTTTACT2058
    IleLeuThrGlyGlnAsnPheThrSerGluSerLysValValPheThr
    600605610
    GAGAAGACCACAGATGGACAGCAAATTTGGGAGATGGAAGCCACGGTG2106
    GluLysThrThrAspGlyGlnGlnIleTrpGluMetGluAlaThrVal
    615620625
    GATAAGGACAAGAGCCAGCCCAACATGCTTTTTGTTGAGATCCCTGAA2154
    AspLysAspLysSerGlnProAsnMetLeuPheValGluIleProGlu
    630635640
    TATCGGAACAAGCATATCCGCACACCTGTAAAAGTGAACTTCTACGTC2202
    TyrArgAsnLysHisIleArgThrProValLysValAsnPheTyrVal
    645650655660
    ATCAATGGGAAGAGAAAACGAAGTCAGCCTCAGCACTTTACCTACCAC2250
    IleAsnGlyLysArgLysArgSerGlnProGlnHisPheThrTyrHis
    665670675
    CCAGTCCCAGCCATCAAGACGGAGCCCACGGATGAATATGACCCCACT2298
    ProValProAlaIleLysThrGluProThrAspGluTyrAspProThr
    680685690
    CTGATCTGCAGCCCCACCCATGGAGGCCTGGGGAGCCAGCCTTACTAC2346
    LeuIleCysSerProThrHisGlyGlyLeuGlySerGlnProTyrTyr
    695700705
    CCCCAGCACCCGATGGTGGCCGAGTCCCCCTCCTGCCTCGTGGCCACC2394
    ProGlnHisProMetValAlaGluSerProSerCysLeuValAlaThr
    710715720
    ATGGCTCCCTGCCAGCAGTTCCGCACGGGGCTCTCATCCCCTGACGCC2442
    MetAlaProCysGlnGlnPheArgThrGlyLeuSerSerProAspAla
    725730735740
    CGCTACCAGCAACAGAACCCAGCGGCCGTACTCTACCAGCGGAGCAAG2490
    ArgTyrGlnGlnGlnAsnProAlaAlaValLeuTyrGlnArgSerLys
    745750755
    AGCCTGAGCCCCAGCCTGCTGGGCTATCAGCAGCCGGCCCTCATGGCC2538
    SerLeuSerProSerLeuLeuGlyTyrGlnGlnProAlaLeuMetAla
    760765770
    GCCCCGCTGTCCCTTGCGGACGCTCACCGCTCTGTGCTGGTGCACGCC2586
    AlaProLeuSerLeuAlaAspAlaHisArgSerValLeuValHisAla
    775780785
    GGCTCCCAGGGCCAGAGCTCAGCCCTGCTCCACCCCTCTCCGACCAAC2634
    GlySerGlnGlyGlnSerSerAlaLeuLeuHisProSerProThrAsn
    790795800
    CAGCAGGCCTCGCCTGTGATCCACTACTCACCCACCAACCAGCAGCTG2682
    GlnGlnAlaSerProValIleHisTyrSerProThrAsnGlnGlnLeu
    805810815820
    CGCTGCGGAAGCCACCAGGAGTTCCAGCACATCATGTACTGCGAGAAT2730
    ArgCysGlySerHisGlnGluPheGlnHisIleMetTyrCysGluAsn
    825830835
    TTCGCACCAGGCACCACCAGACCTGGCCCGCCCCCGGTCAGTCAAGGT2778
    PheAlaProGlyThrThrArgProGlyProProProValSerGlnGly
    840845850
    CAGAGGCTGAGCCCGGGTTCCTACCCCACAGTCATTCAGCAGCAGAAT2826
    GlnArgLeuSerProGlySerTyrProThrValIleGlnGlnGlnAsn
    855860865
    GCCACGAGCCAAAGAGCCGCCAAAAACGGACCCCCGGTCAGTGACCAA2874
    AlaThrSerGlnArgAlaAlaLysAsnGlyProProValSerAspGln
    870875880
    AAGGAAGTATTACCTGCGGGGGTGACCATTAAACAGGAGCAGAACTTG2922
    LysGluValLeuProAlaGlyValThrIleLysGlnGluGlnAsnLeu
    885890895900
    GACCAGACCTACTTGGATGATGAGCTGATAGACACACACCTTAGCTGG2970
    AspGlnThrTyrLeuAspAspGluLeuIleAspThrHisLeuSerTrp
    905910915
    ATACAAAACATATTATGAAACAGAATGACTGTGATCTTTGATCCGAG3017
    IleGlnAsnIleLeu
    920
    AAATCAAAGTTAAAGTTAATGAAATTATCAGGAAGGAGTTTTCAGGACCTCCTGCCAGAA3077
    ATCAGACGTAAAAGAAGCCATTATAGCAAGACACCTTCTGTATCTGACCCCTCGGAGCCC3137
    TCCACAGCCCCTCACCTTCTGTCTCCTTTCATGTTCATCTCCCAGCCCGGAGTCCACACG3197
    CGGATCAATGTATGGGCACTAAGCGGACTCTCACTTAAGGAGCTCGCCACCTCCCTCTAA3257
    ACACCAGAGAGAACTCTTCTTTTCGGTTTATGTTTTAAATCCCAGAGAGCATCCTGGTTG3317
    ATCTTAATGGTGTTCCGTCCAAATAGTAAGCACCTGCTGACCAAAAGCACATTCTACATG3377
    AGACAGGACACTGGAACTCTCCTGAGAACAGAGTGACTGGAGCTTGGGGGGATGGACGGG3437
    GGACAGAAGATGTGGGCACTGTGATTAAACCCCAGCCCTTG3478
    (2) INFORMATION FOR SEQ ID NO:2:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 921 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2:
    MetAsnAlaProGluArgGlnProGlnProAspGlyGlyAspAlaPro
    151015
    GlyHisGluProGlyGlySerProGlnAspGluLeuAspPheSerIle
    202530
    LeuPheAspTyrGluTyrLeuAsnProAsnGluGluGluProAsnAla
    354045
    HisLysValAlaSerProProSerGlyProAlaTyrProAspAspVal
    505560
    LeuAspTyrGlyLeuLysProTyrSerProLeuAlaSerLeuSerGly
    65707580
    GluProProGlyArgPheGlyGluProAspArgValGlyProGlnLys
    859095
    PheLeuSerAlaAlaLysProAlaGlyAlaSerGlyLeuSerProArg
    100105110
    IleGluIleThrProSerHisGluLeuIleGlnAlaValGlyProLeu
    115120125
    ArgMetArgAspAlaGlyLeuLeuValGluGlnProProLeuAlaGly
    130135140
    ValAlaAlaSerProArgPheThrLeuProValProGlyPheGluGly
    145150155160
    TyrArgGluProLeuCysLeuSerProAlaSerSerGlySerSerAla
    165170175
    SerPheIleSerAspThrPheSerProTyrThrSerProCysValSer
    180185190
    ProAsnAsnGlyGlyProAspAspLeuCysProGlnPheGlnAsnIle
    195200205
    ProAlaHisTyrSerProArgThrSerProIleMetSerProArgThr
    210215220
    SerLeuAlaGluAspSerCysLeuGlyArgHisSerProValProArg
    225230235240
    ProAlaSerArgSerSerSerProGlyAlaLysArgArgHisSerCys
    245250255
    AlaGluAlaLeuValAlaLeuProProGlyAlaSerProGlnArgSer
    260265270
    ArgSerProSerProGlnProSerSerHisValAlaProGlnAspHis
    275280285
    GlySerProAlaGlyTyrProProValAlaGlySerAlaValIleMet
    290295300
    AspAlaLeuAsnSerLeuAlaThrAspSerProCysGlyIleProPro
    305310315320
    LysMetTrpLysThrSerProAspProSerProValSerAlaAlaPro
    325330335
    SerLysAlaGlyLeuProArgHisIleTyrProAlaValGluPheLeu
    340345350
    GlyProCysGluGlnGlyGluArgArgAsnSerAlaProGluSerIle
    355360365



    LeuLeuValProProThrTrpProLysProLeuValProAlaIlePro
    370375380
    IleCysSerIleProValThrAlaSerLeuProProLeuGluTrpPro
    385390395400
    LeuSerSerGlnSerGlySerTyrGluLeuArgIleGluValGlnPro
    405410415
    LysProHisHisArgAlaHisTyrGluThrGluGlySerArgGlyAla
    420425430
    ValLysAlaProThrGlyGlyHisProValValGlnLeuHisGlyTyr
    435440445
    MetGluAsnLysProLeuGlyLeuGlnIlePheIleGlyThrAlaAsp
    450455460
    GluArgIleLeuLysProHisAlaPheTyrGlnValHisArgIleThr
    465470475480
    GlyLysThrValThrThrThrSerTyrGluLysIleValGlyAsnThr
    485490495
    LysValLeuGluIleProLeuGluProLysAsnAsnMetArgAlaThr
    500505510
    IleAspCysAlaGlyIleLeuLysLeuArgAsnAlaAspIleGluLeu
    515520525
    ArgLysGlyGluThrAspIleGlyArgLysAsnThrArgValArgLeu
    530535540
    ValPheArgValHisIleProGluSerSerGlyArgIleValSerLeu
    545550555560
    GlnThrAlaSerAsnProIleGluCysSerGlnArgSerAlaHisGlu
    565570575
    LeuProMetValGluArgGlnAspThrAspSerCysLeuValTyrGly
    580585590
    GlyGlnGlnMetIleLeuThrGlyGlnAsnPheThrSerGluSerLys
    595600605
    ValValPheThrGluLysThrThrAspGlyGlnGlnIleTrpGluMet
    610615620
    GluAlaThrValAspLysAspLysSerGlnProAsnMetLeuPheVal
    625630635640
    GluIleProGluTyrArgAsnLysHisIleArgThrProValLysVal
    645650655
    AsnPheTyrValIleAsnGlyLysArgLysArgSerGlnProGlnHis
    660665670
    PheThrTyrHisProValProAlaIleLysThrGluProThrAspGlu
    675680685
    TyrAspProThrLeuIleCysSerProThrHisGlyGlyLeuGlySer
    690695700
    GlnProTyrTyrProGlnHisProMetValAlaGluSerProSerCys
    705710715720
    LeuValAlaThrMetAlaProCysGlnGlnPheArgThrGlyLeuSer
    725730735
    SerProAspAlaArgTyrGlnGlnGlnAsnProAlaAlaValLeuTyr
    740745750
    GlnArgSerLysSerLeuSerProSerLeuLeuGlyTyrGlnGlnPro
    755760765
    AlaLeuMetAlaAlaProLeuSerLeuAlaAspAlaHisArgSerVal
    770775780
    LeuValHisAlaGlySerGlnGlyGlnSerSerAlaLeuLeuHisPro
    785790795800
    SerProThrAsnGlnGlnAlaSerProValIleHisTyrSerProThr
    805810815
    AsnGlnGlnLeuArgCysGlySerHisGlnGluPheGlnHisIleMet
    820825830
    TyrCysGluAsnPheAlaProGlyThrThrArgProGlyProProPro
    835840845
    ValSerGlnGlyGlnArgLeuSerProGlySerTyrProThrValIle
    850855860
    GlnGlnGlnAsnAlaThrSerGlnArgAlaAlaLysAsnGlyProPro
    865870875880
    ValSerAspGlnLysGluValLeuProAlaGlyValThrIleLysGln
    885890895
    GluGlnAsnLeuAspGlnThrTyrLeuAspAspGluLeuIleAspThr
    900905910
    HisLeuSerTrpIleGlnAsnIleLeu
    915920
    (2) INFORMATION FOR SEQ ID NO:3:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 2743 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: CDS
    (B) LOCATION: 240..2390
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3:
    GAATTCCGCAGGGCGCGGGCACCGGGGCGCGGGCAGGGCTCGGAGCCACCGCGCAGGTCC60
    TAGGGCCGCGGCCGGGCCCCGCCACGCGCGCACACGCCCCTCGATGACTTTCCTCCGGGG120
    CGCGCGGCGCTGAGCCCGGGGCGAGGGCTGTCTTCCCGGAGACCCGACCCCGGCAGCGCG180
    GGGCGGCCACTTCTCCTGTGCCTCCGCCCGCTGCTCCACTCCCCGCCGCCGCCGCGCGG239
    ATGCCAAGCACCAGCTTTCCAGTCCCTTCCAAGTTTCCACTTGGCCCT287
    MetProSerThrSerPheProValProSerLysPheProLeuGlyPro
    925930935
    GCGGCTGCGGTCTTCGGGAGAGGAGAAACTTTGGGGCCCGCGCCGCGC335
    AlaAlaAlaValPheGlyArgGlyGluThrLeuGlyProAlaProArg
    940945950
    GCCGGCGGCACCATGAAGTCAGCGGAGGAAGAACACTATGGCTATGCA383
    AlaGlyGlyThrMetLysSerAlaGluGluGluHisTyrGlyTyrAla
    955960965
    TCCTCCAACGTCAGCCCCGCCCTGCCGCTCCCCACGGCGCACTCCACC431
    SerSerAsnValSerProAlaLeuProLeuProThrAlaHisSerThr
    970975980985
    CTGCCGGCCCCGTGCCACAACCTTCAGACCTCCACACCGGGCATCATC479
    LeuProAlaProCysHisAsnLeuGlnThrSerThrProGlyIleIle
    9909951000
    CCGCCGGCGGATCACCCCTCGGGGTACGGAGCAGCTTTGGACGGTGGG527
    ProProAlaAspHisProSerGlyTyrGlyAlaAlaLeuAspGlyGly
    100510101015
    CCCGCGGGCTACTTCCTCTCCTCCGGCCACACCAGGCCTGATGGGGCC575
    ProAlaGlyTyrPheLeuSerSerGlyHisThrArgProAspGlyAla
    102010251030
    CCTGCCCTGGAGAGTCCTCGCATCGAGATAACCTCGTGCTTGGGCCTG623
    ProAlaLeuGluSerProArgIleGluIleThrSerCysLeuGlyLeu
    103510401045
    TACCACAACAATAACCAGTTTTTCCACGATGTGGAGGTGGAAGACGTC671
    TyrHisAsnAsnAsnGlnPhePheHisAspValGluValGluAspVal
    1050105510601065
    CTCCCTAGCTCCAAACGGTCCCCCTCCACGGCCACGCTGAGTCTGCCC719
    LeuProSerSerLysArgSerProSerThrAlaThrLeuSerLeuPro
    107010751080
    AGCCTGGAGGCCTACAGAGACCCCTCGTGCCTGAGCCCGGCCAGCAGC767
    SerLeuGluAlaTyrArgAspProSerCysLeuSerProAlaSerSer
    108510901095
    CTGTCCTCCCGGAGCTGCAACTCAGAGGCCTCCTCCTACGAGTCCAAC815
    LeuSerSerArgSerCysAsnSerGluAlaSerSerTyrGluSerAsn
    110011051110
    TACTCGTACCCGTACGCGTCCCCCCAGACGTCGCCATGGCAGTCTCCC863
    TyrSerTyrProTyrAlaSerProGlnThrSerProTrpGlnSerPro
    111511201125
    TGCGTGTCTCCCAAGACCACGGACCCCGAGGAGGGCTTTCCCCGCGGG911
    CysValSerProLysThrThrAspProGluGluGlyPheProArgGly
    1130113511401145
    CTGGGGGCCTGCACACTGCTGGGTTCCCCGCAGCACTCCCCCTCCACC959
    LeuGlyAlaCysThrLeuLeuGlySerProGlnHisSerProSerThr
    115011551160
    TCGCCCCGCGCCAGCGTCACTGAGGAGAGCTGGCTGGGTGCCCGCTCC1007
    SerProArgAlaSerValThrGluGluSerTrpLeuGlyAlaArgSer
    116511701175
    TCCAGACCCGCGTCCCCTTGCAACAAGAGGAAGTACAGCCTCAACGGC1055
    SerArgProAlaSerProCysAsnLysArgLysTyrSerLeuAsnGly
    118011851190
    CGGCAGCCGCCCTACTCACCCCACCACTCGCCCACGCCGTCCCCGCAC1103
    ArgGlnProProTyrSerProHisHisSerProThrProSerProHis
    119512001205
    GGCTCCCCGCGGGTCAGCGTGACCGACGACTCGTGGTTGGGCAACACC1151
    GlySerProArgValSerValThrAspAspSerTrpLeuGlyAsnThr
    1210121512201225
    ACCCAGTACACCAGCTCGGCCATCGTGGCCGCCATCAACGCGCTGACC1199
    ThrGlnTyrThrSerSerAlaIleValAlaAlaIleAsnAlaLeuThr
    123012351240
    ACCGACAGCAGCCTGGACCTGGGAGATGGCGTCCCTGTCAAGTCCCGC1247
    ThrAspSerSerLeuAspLeuGlyAspGlyValProValLysSerArg
    124512501255
    AAGACCACCCTGGAGCAGCCGCCCTCAGTGGCGCTCAAGGTGGAGCCC1295
    LysThrThrLeuGluGlnProProSerValAlaLeuLysValGluPro
    126012651270
    GTCGGGGAGGACCTGGGCAGCCCCCCGCCCCCGGCCGACTTCGCGCCC1343
    ValGlyGluAspLeuGlySerProProProProAlaAspPheAlaPro
    127512801285
    GAAGACTACTCCTCTTTCCAGCACATCAGGAAGGGCGGCTTCTGCGAC1391
    GluAspTyrSerSerPheGlnHisIleArgLysGlyGlyPheCysAsp
    1290129513001305
    CAGTACCTGGCGGTGCCGCAGCACCCCTACCAGTGGGCGAAGCCCAAG1439
    GlnTyrLeuAlaValProGlnHisProTyrGlnTrpAlaLysProLys
    131013151320
    CCCCTGTCCCCTACGTCCTACATGAGCCCGACCCTGCCCGCCCTGGAC1487
    ProLeuSerProThrSerTyrMetSerProThrLeuProAlaLeuAsp
    132513301335
    TGGCAGCTGCCGTCCCACTCAGGCCCGTATGAGCTTCGGATTGAGGTG1535
    TrpGlnLeuProSerHisSerGlyProTyrGluLeuArgIleGluVal
    134013451350
    CAGCCCAAGTCCCACCACCGAGCCCACTACGAGACGGAGGGCAGCCGG1583
    GlnProLysSerHisHisArgAlaHisTyrGluThrGluGlySerArg
    135513601365
    GGGGCCGTGAAGGCGTCGGCCGGAGGACACCCCATCGTGCAGCTGCAT1631
    GlyAlaValLysAlaSerAlaGlyGlyHisProIleValGlnLeuHis
    1370137513801385
    GGCTACTTGGAGAATGAGCCGCTGATGCTGCAGCTTTTCATTGGGACG1679
    GlyTyrLeuGluAsnGluProLeuMetLeuGlnLeuPheIleGlyThr
    139013951400
    GCGGACGACCGCCTGCTGCGCCCGCACGCCTTCTACCAGGTGCACCGC1727
    AlaAspAspArgLeuLeuArgProHisAlaPheTyrGlnValHisArg
    140514101415
    ATCACAGGGAAGACCGTGTCCACCACCAGCCACGAGGCTATCCTCTCC1775
    IleThrGlyLysThrValSerThrThrSerHisGluAlaIleLeuSer
    142014251430
    AACACCAAAGTCCTGGAGATCCCACTCCTGCCGGAGAACAGCATGCGA1823
    AsnThrLysValLeuGluIleProLeuLeuProGluAsnSerMetArg
    143514401445
    GCCGTCATTGACTGTGCCGGAATCCTGAAACTCAGAAACTCCGACATT1871
    AlaValIleAspCysAlaGlyIleLeuLysLeuArgAsnSerAspIle
    1450145514601465
    GAACTTCGGAAAGGAGAGACGGACATCGGGAGGAAGAACACACGGGTA1919
    GluLeuArgLysGlyGluThrAspIleGlyArgLysAsnThrArgVal
    147014751480
    CGGCTGGTGTTCCGCGTTCACGTCCCGCAACCCAGCGGCCGCACGCTG1967
    ArgLeuValPheArgValHisValProGlnProSerGlyArgThrLeu
    148514901495
    TCCCTGCAGGTGGCCTCCAACCCCATCGAATGCTCCCAGCGCTCAGCT2015
    SerLeuGlnValAlaSerAsnProIleGluCysSerGlnArgSerAla
    150015051510
    CAGGAGCTGCCTCTGGTGGAGAAGCAGAGCACGGACAGCTATCCGGTC2063
    GlnGluLeuProLeuValGluLysGlnSerThrAspSerTyrProVal
    151515201525
    GTGGGCGGGAAGAAGATGGTCCTGTCTGGCCACAACTTCCTGCAGGAC2111
    ValGlyGlyLysLysMetValLeuSerGlyHisAsnPheLeuGlnAsp
    1530153515401545
    TCCAAGGTCATTTTCGTGGAGAAAGCCCCAGATGGCCACCATGTCTGG2159
    SerLysValIlePheValGluLysAlaProAspGlyHisHisValTrp
    155015551560
    GAGATGGAAGCGAAAACTGACCGGGACCTGTGCAAGCCGAATTCTCTG2207
    GluMetGluAlaLysThrAspArgAspLeuCysLysProAsnSerLeu
    156515701575
    GTGGTTGAGATCCCGCCATTTCGGAATCAGAGGATAACCAGCCCCGTT2255
    ValValGluIleProProPheArgAsnGlnArgIleThrSerProVal
    158015851590
    CACGTCAGTTTCTACGTCTGCAACGGGAAGAGAAAGCGAAGCCAGTAC2303
    HisValSerPheTyrValCysAsnGlyLysArgLysArgSerGlnTyr
    159516001605
    CAGCGTTTCACCTACCTTCCCGCCAACGGTAACGCCATCTTTCTAACC2351
    GlnArgPheThrTyrLeuProAlaAsnGlyAsnAlaIlePheLeuThr
    1610161516201625
    GTAAGCCGTGAACATGAGCGCGTGGGGTGCTTTTTCTAAAGACGCAGAA2400
    ValSerArgGluHisGluArgValGlyCysPhePhe*
    16301635
    ACGACGTCGCCGTAAAGCAGCGTGGCGTGTTGCACATTTAACTGTGTGATGTCCCGTTAG2460
    TGAGACCGAGCCATCGATGCCCTGAAAAGGAAAGGAAAAGGGAAGCTTCGGATGCATTTT2520
    CCTTGATCCCTGTTGGGGGTGGGGGGCGGGGGTTGCATACTCAGATAGTCACGGTTATTT2580
    TGCTTCTTGCGAATGTATAACAGCCAAGGGGAAAACATGGCTCTTCTGCTCCAAAAAACT2640
    GAGGGGGTCCTGGTGTGCATTTGCACCCTAAAGCTGCTTACGGTGAAAAGGCAAATAGGT2700
    ATAGCTATTTTGCAGGCACCTTTAGGAATAAACTTTGCTTTTA2743
    (2) INFORMATION FOR SEQ ID NO:4:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 716 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4:
    MetProSerThrSerPheProValProSerLysPheProLeuGlyPro
    151015
    AlaAlaAlaValPheGlyArgGlyGluThrLeuGlyProAlaProArg
    202530
    AlaGlyGlyThrMetLysSerAlaGluGluGluHisTyrGlyTyrAla
    354045
    SerSerAsnValSerProAlaLeuProLeuProThrAlaHisSerThr
    505560
    LeuProAlaProCysHisAsnLeuGlnThrSerThrProGlyIleIle
    65707580
    ProProAlaAspHisProSerGlyTyrGlyAlaAlaLeuAspGlyGly
    859095
    ProAlaGlyTyrPheLeuSerSerGlyHisThrArgProAspGlyAla
    100105110
    ProAlaLeuGluSerProArgIleGluIleThrSerCysLeuGlyLeu
    115120125
    TyrHisAsnAsnAsnGlnPhePheHisAspValGluValGluAspVal
    130135140
    LeuProSerSerLysArgSerProSerThrAlaThrLeuSerLeuPro
    145150155160
    SerLeuGluAlaTyrArgAspProSerCysLeuSerProAlaSerSer
    165170175



    LeuSerSerArgSerCysAsnSerGluAlaSerSerTyrGluSerAsn
    180185190
    TyrSerTyrProTyrAlaSerProGlnThrSerProTrpGlnSerPro
    195200205
    CysValSerProLysThrThrAspProGluGluGlyPheProArgGly
    210215220
    LeuGlyAlaCysThrLeuLeuGlySerProGlnHisSerProSerThr
    225230235240
    SerProArgAlaSerValThrGluGluSerTrpLeuGlyAlaArgSer
    245250255
    SerArgProAlaSerProCysAsnLysArgLysTyrSerLeuAsnGly
    260265270
    ArgGlnProProTyrSerProHisHisSerProThrProSerProHis
    275280285
    GlySerProArgValSerValThrAspAspSerTrpLeuGlyAsnThr
    290295300
    ThrGlnTyrThrSerSerAlaIleValAlaAlaIleAsnAlaLeuThr
    305310315320
    ThrAspSerSerLeuAspLeuGlyAspGlyValProValLysSerArg
    325330335
    LysThrThrLeuGluGlnProProSerValAlaLeuLysValGluPro
    340345350
    ValGlyGluAspLeuGlySerProProProProAlaAspPheAlaPro
    355360365
    GluAspTyrSerSerPheGlnHisIleArgLysGlyGlyPheCysAsp
    370375380
    GlnTyrLeuAlaValProGlnHisProTyrGlnTrpAlaLysProLys
    385390395400
    ProLeuSerProThrSerTyrMetSerProThrLeuProAlaLeuAsp
    405410415
    TrpGlnLeuProSerHisSerGlyProTyrGluLeuArgIleGluVal
    420425430
    GlnProLysSerHisHisArgAlaHisTyrGluThrGluGlySerArg
    435440445
    GlyAlaValLysAlaSerAlaGlyGlyHisProIleValGlnLeuHis
    450455460
    GlyTyrLeuGluAsnGluProLeuMetLeuGlnLeuPheIleGlyThr
    465470475480
    AlaAspAspArgLeuLeuArgProHisAlaPheTyrGlnValHisArg
    485490495
    IleThrGlyLysThrValSerThrThrSerHisGluAlaIleLeuSer
    500505510
    AsnThrLysValLeuGluIleProLeuLeuProGluAsnSerMetArg
    515520525
    AlaValIleAspCysAlaGlyIleLeuLysLeuArgAsnSerAspIle
    530535540
    GluLeuArgLysGlyGluThrAspIleGlyArgLysAsnThrArgVal
    545550555560
    ArgLeuValPheArgValHisValProGlnProSerGlyArgThrLeu
    565570575
    SerLeuGlnValAlaSerAsnProIleGluCysSerGlnArgSerAla
    580585590
    GlnGluLeuProLeuValGluLysGlnSerThrAspSerTyrProVal
    595600605
    ValGlyGlyLysLysMetValLeuSerGlyHisAsnPheLeuGlnAsp
    610615620
    SerLysValIlePheValGluLysAlaProAspGlyHisHisValTrp
    625630635640
    GluMetGluAlaLysThrAspArgAspLeuCysLysProAsnSerLeu
    645650655
    ValValGluIleProProPheArgAsnGlnArgIleThrSerProVal
    660665670
    HisValSerPheTyrValCysAsnGlyLysArgLysArgSerGlnTyr
    675680685
    GlnArgPheThrTyrLeuProAlaAsnGlyAsnAlaIlePheLeuThr
    690695700
    ValSerArgGluHisGluArgValGlyCysPhePhe
    705710715
    (2) INFORMATION FOR SEQ ID NO:5:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 2881 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: CDS
    (B) LOCATION: 142..2850
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5:
    GCTTCTGGAGGGAGGCGGCAGCGACGGAGGAGGGGGCTTCTCAGAGAAAGGGAGGGAGGG60
    AGCCACCCGGGTGAAGATACAGCAGCCTCCTGAACTCCCCCCTCCCACCCAGGCCGGGAC120
    CTGGGGGCTCCTGCCGGATCCATGGGGGCGGCCAGCTGCGAGGATGAGGAG171
    MetGlyAlaAlaSerCysGluAspGluGlu
    720725
    CTGGAATTTAAGCTGGTGTTCGGGGAGGAAAAGGAGGCCCCCCCGCTG219
    LeuGluPheLysLeuValPheGlyGluGluLysGluAlaProProLeu
    730735740
    GGCGCGGGGGGATTGGGGGAAGAACTGGACTCAGAGGATGCCCCGCCA267
    GlyAlaGlyGlyLeuGlyGluGluLeuAspSerGluAspAlaProPro
    745750755
    TGCTGCCGTCTGGCCTTGGGAGAGCCCCCTCCCTATGGCGCTGCACCT315
    CysCysArgLeuAlaLeuGlyGluProProProTyrGlyAlaAlaPro
    760765770775
    ATCGGTATTCCCCGACCTCCACCCCCTCGGCCTGGCATGCATTCGCCA363
    IleGlyIleProArgProProProProArgProGlyMetHisSerPro
    780785790
    CCGCCGCGACCAGCCCCCTCACCTGGCACCTGGGAGAGCCAGCCCGCC411
    ProProArgProAlaProSerProGlyThrTrpGluSerGlnProAla
    795800805
    AGGTCGGTGAGGCTGGGAGGACCAGGAGGGGGTGCTGGGGGTGCTGGG459
    ArgSerValArgLeuGlyGlyProGlyGlyGlyAlaGlyGlyAlaGly
    810815820
    GGTGGCCGTGTTCTCGAGTGTCCCAGCATCCGCATCACCTCCATCTCT507
    GlyGlyArgValLeuGluCysProSerIleArgIleThrSerIleSer
    825830835
    CCCACGCCGGAGCCGCCAGCAGCGCTGGAGGACAACCCTGATGCCTGG555
    ProThrProGluProProAlaAlaLeuGluAspAsnProAspAlaTrp
    840845850855
    GGGGACGGCTCTCCTAGAGATTACCCCCCACCAGAAGGCTTTGGGGGC603
    GlyAspGlySerProArgAspTyrProProProGluGlyPheGlyGly
    860865870
    TACAGAGAAGCAGGGGCCCAGGGTGGGGGGGCCTTCTTCAGCCCAAGC651
    TyrArgGluAlaGlyAlaGlnGlyGlyGlyAlaPhePheSerProSer
    875880885
    CCTGGCAGCAGCAGCCTGTCCTCGTGGAGCTTCTTCTCCGATGCCTCT699
    ProGlySerSerSerLeuSerSerTrpSerPhePheSerAspAlaSer
    890895900
    GACGAGGCAGCCCTGTATGCAGCCTGCGACGAGGTGGAGTCTGAGCTA747
    AspGluAlaAlaLeuTyrAlaAlaCysAspGluValGluSerGluLeu
    905910915
    AATGAGGCGGCCTCCCGCTTTGGCCTGGGCTCCCCGCTGCCCTCGCCC795
    AsnGluAlaAlaSerArgPheGlyLeuGlySerProLeuProSerPro
    920925930935
    CGGGCCTCCCCTCGGCCATGGACCCCCGAAGATCCCTGGAGCCTGTAT843
    ArgAlaSerProArgProTrpThrProGluAspProTrpSerLeuTyr
    940945950
    GGTCCAAGCCCCGGAGGCCGAGGGCCAGAGGATAGCTGGCTACTCCTC891
    GlyProSerProGlyGlyArgGlyProGluAspSerTrpLeuLeuLeu
    955960965
    AGTGCTCCTGGGCCCACCCCAGCCTCCCCGCGGCCTGCCTCTCCATGT939
    SerAlaProGlyProThrProAlaSerProArgProAlaSerProCys
    970975980
    GGCAAGCGGCGCTATTCCAGCTCGGGAACCCCATCTTCAGCCTCCCCA987
    GlyLysArgArgTyrSerSerSerGlyThrProSerSerAlaSerPro
    985990995
    GCTCTGTCCCGCCGTGGCAGCCTGGGGGAAGAGGGGTCTGAGCCACCT1035
    AlaLeuSerArgArgGlySerLeuGlyGluGluGlySerGluProPro
    1000100510101015
    CCACCACCCCCATTGCCTCTGGCCCGGGACCCGGGCTCCCCTGGTCCC1083
    ProProProProLeuProLeuAlaArgAspProGlySerProGlyPro
    102010251030
    TTTGACTATGTGGGGGCCCCACCAGCTGAGAGCATCCCTCAGAAGACA1131
    PheAspTyrValGlyAlaProProAlaGluSerIleProGlnLysThr
    103510401045
    CGGCGGACTTCCAGCGAGCAGGCAGTGGCTCTGCCTCGGTCTGAGGAG1179
    ArgArgThrSerSerGluGlnAlaValAlaLeuProArgSerGluGlu
    105010551060
    CCTGCCTCATGCAATGGGAAGCTGCCCTTGGGAGCAGAGGAGTCTGTG1227
    ProAlaSerCysAsnGlyLysLeuProLeuGlyAlaGluGluSerVal
    106510701075
    GCTCCTCCAGGAGGTTCCCGGAAGGAGGTGGCTGGCATGGACTACCTG1275
    AlaProProGlyGlySerArgLysGluValAlaGlyMetAspTyrLeu
    1080108510901095
    GCAGTGCCCTCCCCACTCGCTTGGTCCAAGGCCCGGATTGGGGGACAC1323
    AlaValProSerProLeuAlaTrpSerLysAlaArgIleGlyGlyHis
    110011051110
    AGCCCTATCTTCAGGACCTCTGCCCTACCCCCACTGGACTGGCCTCTG1371
    SerProIlePheArgThrSerAlaLeuProProLeuAspTrpProLeu
    111511201125
    CCCAGCCAATATGAGCAGCTGGAGCTGAGGATCGAGGTACAGCCTAGA1419
    ProSerGlnTyrGluGlnLeuGluLeuArgIleGluValGlnProArg
    113011351140
    GCCCACCACCGGGCCCACTATGAGACAGAAGGCAGCCGTGGAGCTGTC1467
    AlaHisHisArgAlaHisTyrGluThrGluGlySerArgGlyAlaVal
    114511501155
    AAAGCTGCCCCTGGCGGTCACCCCGTAGTCAAGCTCCTAGGCTACAGT1515
    LysAlaAlaProGlyGlyHisProValValLysLeuLeuGlyTyrSer
    1160116511701175
    GAGAAGCCACTGACCCTACAGATGTTCATCGGCACTGCAGATGAAAGG1563
    GluLysProLeuThrLeuGlnMetPheIleGlyThrAlaAspGluArg
    118011851190
    AACCTGCGGCCTCATGCCTTCTATCAGGTGCACCGTATCACAGGCAAG1611
    AsnLeuArgProHisAlaPheTyrGlnValHisArgIleThrGlyLys
    119512001205
    ATGGTGGCCACGGCCAGCTATGAAGCCGTAGTCAGTGGCACCAAGGTG1659
    MetValAlaThrAlaSerTyrGluAlaValValSerGlyThrLysVal
    121012151220
    TTGGAGATGACTCTGCTGCCTGAGAACAACATGGCGGCCAACATTGAC1707
    LeuGluMetThrLeuLeuProGluAsnAsnMetAlaAlaAsnIleAsp
    122512301235
    TGCGCGGGAATCCTGAAGCTTCGGAATTCAGACATTGAGCTTCGGAAG1755
    CysAlaGlyIleLeuLysLeuArgAsnSerAspIleGluLeuArgLys
    1240124512501255
    GGTGAGACGGACATCGGGCGCAAAAACACACGTGTACGGCTGGTGTTC1803
    GlyGluThrAspIleGlyArgLysAsnThrArgValArgLeuValPhe
    126012651270
    CGGGTACACGTGCCCCAGGGCGGCGGGAAGGTCGTCTCAGTACAGGCA1851
    ArgValHisValProGlnGlyGlyGlyLysValValSerValGlnAla
    127512801285
    GCATCGGTGCCCATCGAGTGCTCCCAGCGCTCAGCCCAGGAGCTGCCC1899
    AlaSerValProIleGluCysSerGlnArgSerAlaGlnGluLeuPro
    129012951300
    CAGGTGGAGGCCTACAGCCCCAGTGCCTGCTCTGTGAGAGGAGGCGAG1947
    GlnValGluAlaTyrSerProSerAlaCysSerValArgGlyGlyGlu
    130513101315
    GAACTGGTACTGACCGGCTCCAACTTCCTGCCAGACTCCAAGGTGGTG1995
    GluLeuValLeuThrGlySerAsnPheLeuProAspSerLysValVal
    1320132513301335
    TTCATTGAGAGGGGTCCTGATGGGAAGCTGCAATGGGAGGAGGAGGCC2043
    PheIleGluArgGlyProAspGlyLysLeuGlnTrpGluGluGluAla
    134013451350
    ACAGTGAACCGACTGCAGAGCAACGAGGTGACGCTGACCCTGACTGTC2091
    ThrValAsnArgLeuGlnSerAsnGluValThrLeuThrLeuThrVal
    135513601365
    CCCGAGTACAGCAACAAGAGGGTTTCCCGGCCAGTCCAGGTCTACTTT2139
    ProGluTyrSerAsnLysArgValSerArgProValGlnValTyrPhe
    137013751380
    TATGTCTCCAATGGGCGGAGGAAACGCAGTCCTACCCAGAGTTTCAGG2187
    TyrValSerAsnGlyArgArgLysArgSerProThrGlnSerPheArg
    138513901395
    TTTCTGCCTGTGATCTGCAAAGAGGAGCCCCTACCGGACTCATCTCTG2235
    PheLeuProValIleCysLysGluGluProLeuProAspSerSerLeu
    1400140514101415
    CGGGGTTTCCCTTCAGCATCGGCAACCCCCTTTGGCACTGACATGGAC2283
    ArgGlyPheProSerAlaSerAlaThrProPheGlyThrAspMetAsp
    142014251430
    TTCTCACCACCCAGGCCCCCCTACCCCTCCTATCCCCATGAAGACCCT2331
    PheSerProProArgProProTyrProSerTyrProHisGluAspPro
    143514401445
    GCTTGCGAAACTCCTTACCTATCAGAAGGCTTCGGCTATGGCATGCCC2379
    AlaCysGluThrProTyrLeuSerGluGlyPheGlyTyrGlyMetPro
    145014551460
    CCTCTGTACCCCCAGACGGGGCCCCCACCATCCTACAGACCGGGCCTG2427
    ProLeuTyrProGlnThrGlyProProProSerTyrArgProGlyLeu
    146514701475
    CGGATGTTCCCTGAGACTAGGGGTACCACAGGTTGTGCCCAACCACCT2475
    ArgMetPheProGluThrArgGlyThrThrGlyCysAlaGlnProPro
    1480148514901495
    GCAGTTTCCTTCCTTCCCCGCCCCTTCCCTAGTGACCCGTATGGAGGG2523
    AlaValSerPheLeuProArgProPheProSerAspProTyrGlyGly
    150015051510
    CGGGGCTCCTCTTTCCCCCTGGGGCTGCCATTCTCTCCGCCAGCCCCC2571
    ArgGlySerSerPheProLeuGlyLeuProPheSerProProAlaPro
    151515201525
    TTTCGGCCGCCTCCTCTTCCTGCATCCCCACCGCTTGAAGGCCCCTTC2619
    PheArgProProProLeuProAlaSerProProLeuGluGlyProPhe
    153015351540
    CCTTCCCAGAGTGATGTGCATCCCCTACCTGCTGAGGGATACAATAAG2667
    ProSerGlnSerAspValHisProLeuProAlaGluGlyTyrAsnLys
    154515501555
    GTAGGGCCAGGCTATGGCCCTGGGGAGGGGGCTCCGGAGCAGGAGAAA2715
    ValGlyProGlyTyrGlyProGlyGluGlyAlaProGluGlnGluLys
    1560156515701575
    TCCAGGGGTGGCTACAGCAGCGGCTTTCGAGACAGTGTCCCTATCCAG2763
    SerArgGlyGlyTyrSerSerGlyPheArgAspSerValProIleGln
    158015851590
    GGTATCACGCTGGAGGAAGTGAGTGAGATCATTGGCCGAGACCTGAGT2811
    GlyIleThrLeuGluGluValSerGluIleIleGlyArgAspLeuSer
    159516001605
    GGCTTCCCTGCACCTCCTGGAGAAGAGCCTCCTGCCTGAACCACGTGAA2860
    GlyPheProAlaProProGlyGluGluProProAla*
    161016151620
    CTGTCATCACCTGGCAACCCC2881
    (2) INFORMATION FOR SEQ ID NO:6:
    (i) SEQUENCE CHARACTERISTICS:



    (A) LENGTH: 902 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6:
    MetGlyAlaAlaSerCysGluAspGluGluLeuGluPheLysLeuVal
    151015
    PheGlyGluGluLysGluAlaProProLeuGlyAlaGlyGlyLeuGly
    202530
    GluGluLeuAspSerGluAspAlaProProCysCysArgLeuAlaLeu
    354045
    GlyGluProProProTyrGlyAlaAlaProIleGlyIleProArgPro
    505560
    ProProProArgProGlyMetHisSerProProProArgProAlaPro
    65707580
    SerProGlyThrTrpGluSerGlnProAlaArgSerValArgLeuGly
    859095
    GlyProGlyGlyGlyAlaGlyGlyAlaGlyGlyGlyArgValLeuGlu
    100105110
    CysProSerIleArgIleThrSerIleSerProThrProGluProPro
    115120125
    AlaAlaLeuGluAspAsnProAspAlaTrpGlyAspGlySerProArg
    130135140
    AspTyrProProProGluGlyPheGlyGlyTyrArgGluAlaGlyAla
    145150155160
    GlnGlyGlyGlyAlaPhePheSerProSerProGlySerSerSerLeu
    165170175
    SerSerTrpSerPhePheSerAspAlaSerAspGluAlaAlaLeuTyr
    180185190
    AlaAlaCysAspGluValGluSerGluLeuAsnGluAlaAlaSerArg
    195200205
    PheGlyLeuGlySerProLeuProSerProArgAlaSerProArgPro
    210215220
    TrpThrProGluAspProTrpSerLeuTyrGlyProSerProGlyGly
    225230235240
    ArgGlyProGluAspSerTrpLeuLeuLeuSerAlaProGlyProThr
    245250255
    ProAlaSerProArgProAlaSerProCysGlyLysArgArgTyrSer
    260265270
    SerSerGlyThrProSerSerAlaSerProAlaLeuSerArgArgGly
    275280285
    SerLeuGlyGluGluGlySerGluProProProProProProLeuPro
    290295300
    LeuAlaArgAspProGlySerProGlyProPheAspTyrValGlyAla
    305310315320
    ProProAlaGluSerIleProGlnLysThrArgArgThrSerSerGlu
    325330335
    GlnAlaValAlaLeuProArgSerGluGluProAlaSerCysAsnGly
    340345350
    LysLeuProLeuGlyAlaGluGluSerValAlaProProGlyGlySer
    355360365
    ArgLysGluValAlaGlyMetAspTyrLeuAlaValProSerProLeu
    370375380
    AlaTrpSerLysAlaArgIleGlyGlyHisSerProIlePheArgThr
    385390395400
    SerAlaLeuProProLeuAspTrpProLeuProSerGlnTyrGluGln
    405410415
    LeuGluLeuArgIleGluValGlnProArgAlaHisHisArgAlaHis
    420425430
    TyrGluThrGluGlySerArgGlyAlaValLysAlaAlaProGlyGly
    435440445
    HisProValValLysLeuLeuGlyTyrSerGluLysProLeuThrLeu
    450455460
    GlnMetPheIleGlyThrAlaAspGluArgAsnLeuArgProHisAla
    465470475480
    PheTyrGlnValHisArgIleThrGlyLysMetValAlaThrAlaSer
    485490495
    TyrGluAlaValValSerGlyThrLysValLeuGluMetThrLeuLeu
    500505510
    ProGluAsnAsnMetAlaAlaAsnIleAspCysAlaGlyIleLeuLys
    515520525
    LeuArgAsnSerAspIleGluLeuArgLysGlyGluThrAspIleGly
    530535540
    ArgLysAsnThrArgValArgLeuValPheArgValHisValProGln
    545550555560
    GlyGlyGlyLysValValSerValGlnAlaAlaSerValProIleGlu
    565570575
    CysSerGlnArgSerAlaGlnGluLeuProGlnValGluAlaTyrSer
    580585590
    ProSerAlaCysSerValArgGlyGlyGluGluLeuValLeuThrGly
    595600605
    SerAsnPheLeuProAspSerLysValValPheIleGluArgGlyPro
    610615620
    AspGlyLysLeuGlnTrpGluGluGluAlaThrValAsnArgLeuGln
    625630635640
    SerAsnGluValThrLeuThrLeuThrValProGluTyrSerAsnLys
    645650655
    ArgValSerArgProValGlnValTyrPheTyrValSerAsnGlyArg
    660665670
    ArgLysArgSerProThrGlnSerPheArgPheLeuProValIleCys
    675680685
    LysGluGluProLeuProAspSerSerLeuArgGlyPheProSerAla
    690695700
    SerAlaThrProPheGlyThrAspMetAspPheSerProProArgPro
    705710715720
    ProTyrProSerTyrProHisGluAspProAlaCysGluThrProTyr
    725730735
    LeuSerGluGlyPheGlyTyrGlyMetProProLeuTyrProGlnThr
    740745750
    GlyProProProSerTyrArgProGlyLeuArgMetPheProGluThr
    755760765
    ArgGlyThrThrGlyCysAlaGlnProProAlaValSerPheLeuPro
    770775780
    ArgProPheProSerAspProTyrGlyGlyArgGlySerSerPhePro
    785790795800
    LeuGlyLeuProPheSerProProAlaProPheArgProProProLeu
    805810815
    ProAlaSerProProLeuGluGlyProPheProSerGlnSerAspVal
    820825830
    HisProLeuProAlaGluGlyTyrAsnLysValGlyProGlyTyrGly
    835840845
    ProGlyGluGlyAlaProGluGlnGluLysSerArgGlyGlyTyrSer
    850855860
    SerGlyPheArgAspSerValProIleGlnGlyIleThrLeuGluGlu
    865870875880
    ValSerGluIleIleGlyArgAspLeuSerGlyPheProAlaProPro
    885890895
    GlyGluGluProProAla
    900
    (2) INFORMATION FOR SEQ ID NO:7:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 2406 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: CDS
    (B) LOCATION: 211..2337
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7:
    CGGCTGCGGTTCCTGGTGCTGCTCGGCGCGCGGCCAGCTTTCGGAACGGAACGCTCGGCG60
    TCGCGGGCCCCGCCCGGAAAGTTTGCCGTGGAGTCGCGACCTCTTGGCCCGCGCGGCCCG120
    GCATGAAGCGGCGTTGAGGAGCTGCTGCCGCCGCTTGCCGCTGCCGCCGCCGCCGCCTGA180
    GGAGGAGCTGCAGCACCCTGGGCCACGCCGATGACTACTGCAAACTGTGGCGCC234
    MetThrThrAlaAsnCysGlyAla
    905910
    CACGACGAGCTCGACTTCAAACTCGTCTTTGGCGAGGACGGGGCGCCG282
    HisAspGluLeuAspPheLysLeuValPheGlyGluAspGlyAlaPro
    915920925
    GCGCCGCCGCCCCCGGGCTCGCGGCCTGCAGATCTTGAGCCAGATGAT330
    AlaProProProProGlySerArgProAlaAspLeuGluProAspAsp
    930935940
    TGTGCATCCATTTACATCTTTAATGTAGATCCACCTCCATCTACTTTA378
    CysAlaSerIleTyrIlePheAsnValAspProProProSerThrLeu
    945950955
    ACCACACCACTTTGCTTACCACATCATGGATTACCGTCTCACTCTTCT426
    ThrThrProLeuCysLeuProHisHisGlyLeuProSerHisSerSer
    960965970975
    GTTTTGTCACCATCGTTTCAGCTCCAAAGTCACAAAAACTATGAAGGA474
    ValLeuSerProSerPheGlnLeuGlnSerHisLysAsnTyrGluGly
    980985990
    ACTTGTGAGATTCCTGAATCTAAATATAGCCCATTAGGTGGTCCCAAA522
    ThrCysGluIleProGluSerLysTyrSerProLeuGlyGlyProLys
    99510001005
    CCCTTTGAGTGCCCAAGTATTCAAATTACATCTATCTCTCCTAACTGT570
    ProPheGluCysProSerIleGlnIleThrSerIleSerProAsnCys
    101010151020
    CATCAAGAATTAGATGCACATGAAGATGACCTACAGATAAATGACCCA618
    HisGlnGluLeuAspAlaHisGluAspAspLeuGlnIleAsnAspPro
    102510301035
    GAACGGGAATTTTTGGAAAGGCCTTCTAGAGATCATCTCTATCTTCCT666
    GluArgGluPheLeuGluArgProSerArgAspHisLeuTyrLeuPro
    1040104510501055
    CTTGAGCCATCCTACCGGGAGTCTTCTCTTAGTCCTAGTCCTGCCAGC714
    LeuGluProSerTyrArgGluSerSerLeuSerProSerProAlaSer
    106010651070
    AGCATCTCTTCTAGGAGTTGGTTCTCTGATGCATCTTCTTGTGAATCG762
    SerIleSerSerArgSerTrpPheSerAspAlaSerSerCysGluSer
    107510801085
    CTTTCACATATTTATGATGATGTGGACTCAGAGTTGAATGAAGCTGCA810
    LeuSerHisIleTyrAspAspValAspSerGluLeuAsnGluAlaAla
    109010951100
    GCCCGATTTACCCTTGGATCCCCTCTGACTTCTCCTGGTGGCTCTCCA858
    AlaArgPheThrLeuGlySerProLeuThrSerProGlyGlySerPro
    110511101115
    GGGGGCTGCCCTGGAGAAGAAACTTGGCATCAACAGTATGGACTTGGA906
    GlyGlyCysProGlyGluGluThrTrpHisGlnGlnTyrGlyLeuGly
    1120112511301135
    CACTCATTATCACCCAGGCAATCTCCTTGCCACTCTCCTAGATCCAGT954
    HisSerLeuSerProArgGlnSerProCysHisSerProArgSerSer
    114011451150
    GTCACTGATGAGAATTGGCTGAGCCCCAGGCCAGCCTCAGGACCCTCA1002
    ValThrAspGluAsnTrpLeuSerProArgProAlaSerGlyProSer
    115511601165
    TCAAGGCCCACATCCCCCTGTGGGAAACGGAGGCACTCCAGTGCTGAA1050
    SerArgProThrSerProCysGlyLysArgArgHisSerSerAlaGlu
    117011751180
    GTTTGTTATGCTGGGTCCCTTTCACCCCATCACTCACCTGTTCCTTCA1098
    ValCysTyrAlaGlySerLeuSerProHisHisSerProValProSer
    118511901195
    CCTGGTCACTCCCCCAGGGGAAGTGTGACAGAAGATACGTGGCTCAAT1146
    ProGlyHisSerProArgGlySerValThrGluAspThrTrpLeuAsn
    1200120512101215
    GCTTCTGTCCATGGTGGGTCAGGCCTTGGCCCTGCAGTTTTTCCATTT1194
    AlaSerValHisGlyGlySerGlyLeuGlyProAlaValPheProPhe
    122012251230
    CAGTACTGTGTAGAGACTGACATCCCTCTCAAAACAAGGAAAACTTCT1242
    GlnTyrCysValGluThrAspIleProLeuLysThrArgLysThrSer
    123512401245
    GAAGATCAAGCTGCCATACTACCAGGAAAATTAGAGCTGTGTTCAGAT1290
    GluAspGlnAlaAlaIleLeuProGlyLysLeuGluLeuCysSerAsp
    125012551260
    GACCAAGGGAGTTTATCACCAGCCCGGGAGACTTCAATAGATGATGGC1338
    AspGlnGlySerLeuSerProAlaArgGluThrSerIleAspAspGly
    126512701275
    CTTGGATCTCAGTATCCTTTAAAGAAAGATTCATGTGGTGATCAGTTT1386
    LeuGlySerGlnTyrProLeuLysLysAspSerCysGlyAspGlnPhe
    1280128512901295
    CTTTCAGTTCCTTCACCCTTTACCTGGAGCAAACCAAAGCCTGGCCAC1434
    LeuSerValProSerProPheThrTrpSerLysProLysProGlyHis
    130013051310
    ACCCCTATATTTCGCACATCTTCATTACCTCCACTAGACTGGCCTTTA1482
    ThrProIlePheArgThrSerSerLeuProProLeuAspTrpProLeu
    131513201325
    CCAGCTCATTTTGGACAATGTGAACTGAAAATAGAAGTGCAACCTAAA1530
    ProAlaHisPheGlyGlnCysGluLeuLysIleGluValGlnProLys
    133013351340
    ACTCATCATCGAGCCCATTATGAAACTGAAGGTAGCCGAGGGGCAGTA1578
    ThrHisHisArgAlaHisTyrGluThrGluGlySerArgGlyAlaVal
    134513501355
    AAAGCATCTACTGGGGGACATCCTGTTGTGAAGCTCCTGGGCTATAAC1626
    LysAlaSerThrGlyGlyHisProValValLysLeuLeuGlyTyrAsn
    1360136513701375
    GAAAAGCCAATAAATCTACAAATGTTTATTGGGACAGCAGATGATCGA1674
    GluLysProIleAsnLeuGlnMetPheIleGlyThrAlaAspAspArg
    138013851390
    TATTTACGACCTCATGCATTTTACCAGGTGCATCGAATCACTGGGAAG1722
    TyrLeuArgProHisAlaPheTyrGlnValHisArgIleThrGlyLys
    139514001405
    ACAGTCGCTACTGCAAGCCAAGAGATAATAATTGCCAGTACAAAAGTT1770
    ThrValAlaThrAlaSerGlnGluIleIleIleAlaSerThrLysVal
    141014151420
    CTGGAAATTCCACTTCTTCCTGAAAATAATATGTCAGCCAGTATTGAT1818
    LeuGluIleProLeuLeuProGluAsnAsnMetSerAlaSerIleAsp
    142514301435
    TGTGCAGGTATTTTGAAACTCCGCAATTCAGATATAGAACTTCGAAAA1866
    CysAlaGlyIleLeuLysLeuArgAsnSerAspIleGluLeuArgLys
    1440144514501455
    GGAGAAACTGATATTGGCAGAAAGAATACTAGAGTACGACTTGTGTTT1914
    GlyGluThrAspIleGlyArgLysAsnThrArgValArgLeuValPhe
    146014651470
    CGTGTACACATCCCACAGCCCAGTGGAAAAGTCCTTTCTCTGCAGATA1962
    ArgValHisIleProGlnProSerGlyLysValLeuSerLeuGlnIle
    147514801485
    GCCTCTATACCCGTTGAGTGCTCCCAGCGGTCTGCTCAAGAACTTCCT2010
    AlaSerIleProValGluCysSerGlnArgSerAlaGlnGluLeuPro
    149014951500
    CATATTGAGAAGTACAGTATCAACAGTTGTTCTGTAAATGGAGGTCAT2058
    HisIleGluLysTyrSerIleAsnSerCysSerValAsnGlyGlyHis
    150515101515
    GAAATGGTTGTGACTGGATCTAATTTTCTTCCAGAATCCAAAATCATT2106
    GluMetValValThrGlySerAsnPheLeuProGluSerLysIleIle
    1520152515301535
    TTTCTTGAAAAAGGACAAGATGGACGACCTCAGTGGGAGGTAGAAGGG2154
    PheLeuGluLysGlyGlnAspGlyArgProGlnTrpGluValGluGly



    154015451550
    AAGATAATCAGGGAAAAATGTCAAGGGGCTCACATTGTCCTTGAAGTT2202
    LysIleIleArgGluLysCysGlnGlyAlaHisIleValLeuGluVal
    155515601565
    CCTCCATATCATAACCCAGCAGTTACAGCTGCAGTGCAGGTGCACTTT2250
    ProProTyrHisAsnProAlaValThrAlaAlaValGlnValHisPhe
    157015751580
    TATCTTTGCAATGGCAAGAGGAAAAAAAGCCAGTCTCAACGTTTTACT2298
    TyrLeuCysAsnGlyLysArgLysLysSerGlnSerGlnArgPheThr
    158515901595
    TATACACCAGGTACGAGGAGTCATGATGGTTTACTATAGAGCTTTCTTT2347
    TyrThrProGlyThrArgSerHisAspGlyLeuLeu*
    160016051610
    CCTAATGAATAAAAAGTTATTTAACGAACAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA2406
    (2) INFORMATION FOR SEQ ID NO:8:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 708 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8:
    MetThrThrAlaAsnCysGlyAlaHisAspGluLeuAspPheLysLeu
    151015
    ValPheGlyGluAspGlyAlaProAlaProProProProGlySerArg
    202530
    ProAlaAspLeuGluProAspAspCysAlaSerIleTyrIlePheAsn
    354045
    ValAspProProProSerThrLeuThrThrProLeuCysLeuProHis
    505560
    HisGlyLeuProSerHisSerSerValLeuSerProSerPheGlnLeu
    65707580
    GlnSerHisLysAsnTyrGluGlyThrCysGluIleProGluSerLys
    859095
    TyrSerProLeuGlyGlyProLysProPheGluCysProSerIleGln
    100105110
    IleThrSerIleSerProAsnCysHisGlnGluLeuAspAlaHisGlu
    115120125
    AspAspLeuGlnIleAsnAspProGluArgGluPheLeuGluArgPro
    130135140
    SerArgAspHisLeuTyrLeuProLeuGluProSerTyrArgGluSer
    145150155160
    SerLeuSerProSerProAlaSerSerIleSerSerArgSerTrpPhe
    165170175
    SerAspAlaSerSerCysGluSerLeuSerHisIleTyrAspAspVal
    180185190
    AspSerGluLeuAsnGluAlaAlaAlaArgPheThrLeuGlySerPro
    195200205
    LeuThrSerProGlyGlySerProGlyGlyCysProGlyGluGluThr
    210215220
    TrpHisGlnGlnTyrGlyLeuGlyHisSerLeuSerProArgGlnSer
    225230235240
    ProCysHisSerProArgSerSerValThrAspGluAsnTrpLeuSer
    245250255
    ProArgProAlaSerGlyProSerSerArgProThrSerProCysGly
    260265270
    LysArgArgHisSerSerAlaGluValCysTyrAlaGlySerLeuSer
    275280285
    ProHisHisSerProValProSerProGlyHisSerProArgGlySer
    290295300
    ValThrGluAspThrTrpLeuAsnAlaSerValHisGlyGlySerGly
    305310315320
    LeuGlyProAlaValPheProPheGlnTyrCysValGluThrAspIle
    325330335
    ProLeuLysThrArgLysThrSerGluAspGlnAlaAlaIleLeuPro
    340345350
    GlyLysLeuGluLeuCysSerAspAspGlnGlySerLeuSerProAla
    355360365
    ArgGluThrSerIleAspAspGlyLeuGlySerGlnTyrProLeuLys
    370375380
    LysAspSerCysGlyAspGlnPheLeuSerValProSerProPheThr
    385390395400
    TrpSerLysProLysProGlyHisThrProIlePheArgThrSerSer
    405410415
    LeuProProLeuAspTrpProLeuProAlaHisPheGlyGlnCysGlu
    420425430
    LeuLysIleGluValGlnProLysThrHisHisArgAlaHisTyrGlu
    435440445
    ThrGluGlySerArgGlyAlaValLysAlaSerThrGlyGlyHisPro
    450455460
    ValValLysLeuLeuGlyTyrAsnGluLysProIleAsnLeuGlnMet
    465470475480
    PheIleGlyThrAlaAspAspArgTyrLeuArgProHisAlaPheTyr
    485490495
    GlnValHisArgIleThrGlyLysThrValAlaThrAlaSerGlnGlu
    500505510
    IleIleIleAlaSerThrLysValLeuGluIleProLeuLeuProGlu
    515520525
    AsnAsnMetSerAlaSerIleAspCysAlaGlyIleLeuLysLeuArg
    530535540
    AsnSerAspIleGluLeuArgLysGlyGluThrAspIleGlyArgLys
    545550555560
    AsnThrArgValArgLeuValPheArgValHisIleProGlnProSer
    565570575
    GlyLysValLeuSerLeuGlnIleAlaSerIleProValGluCysSer
    580585590
    GlnArgSerAlaGlnGluLeuProHisIleGluLysTyrSerIleAsn
    595600605
    SerCysSerValAsnGlyGlyHisGluMetValValThrGlySerAsn
    610615620
    PheLeuProGluSerLysIleIlePheLeuGluLysGlyGlnAspGly
    625630635640
    ArgProGlnTrpGluValGluGlyLysIleIleArgGluLysCysGln
    645650655
    GlyAlaHisIleValLeuGluValProProTyrHisAsnProAlaVal
    660665670
    ThrAlaAlaValGlnValHisPheTyrLeuCysAsnGlyLysArgLys
    675680685
    LysSerGlnSerGlnArgPheThrTyrThrProGlyThrArgSerHis
    690695700
    AspGlyLeuLeu
    705
    (2) INFORMATION FOR SEQ ID NO:09:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 2647 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: CDS
    (B) LOCATION: 211..2427
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:09:
    CGGCTGCGGTTCCTGGTGCTGCTCGGCGCGCGGCCAGCTTTCGGAACGGAACGCTCGGCG60
    TCGCGGGCCCCGCCCGGAAAGTTTGCCGTGGAGTCGCGACCTCTTGGCCCGCGCGGCCCG120
    GCATGAAGCGGCGTTGAGGAGCTGCTGCCGCCGCTTGCCGCTGCCGCCGCCGCCGCCTGA180
    GGAGGAGCTGCAGCACCCTGGGCCACGCCGATGACTACTGCAAACTGTGGCGCC234
    MetThrThrAlaAsnCysGlyAla
    15
    CACGACGAGCTCGACTTCAAACTCGTCTTTGGCGAGGACGGGGCGCCG282
    HisAspGluLeuAspPheLysLeuValPheGlyGluAspGlyAlaPro
    101520
    GCGCCGCCGCCCCCGGGCTCGCGGCCTGCAGATCTTGAGCCAGATGAT330
    AlaProProProProGlySerArgProAlaAspLeuGluProAspAsp
    25303540
    TGTGCATCCATTTACATCTTTAATGTAGATCCACCTCCATCTACTTTA378
    CysAlaSerIleTyrIlePheAsnValAspProProProSerThrLeu
    455055
    ACCACACCACTTTGCTTACCACATCATGGATTACCGTCTCACTCTTCT426
    ThrThrProLeuCysLeuProHisHisGlyLeuProSerHisSerSer
    606570
    GTTTTGTCACCATCGTTTCAGCTCCAAAGTCACAAAAACTATGAAGGA474
    ValLeuSerProSerPheGlnLeuGlnSerHisLysAsnTyrGluGly
    758085
    ACTTGTGAGATTCCTGAATCTAAATATAGCCCATTAGGTGGTCCCAAA522
    ThrCysGluIleProGluSerLysTyrSerProLeuGlyGlyProLys
    9095100
    CCCTTTGAGTGCCCAAGTATTCAAATTACATCTATCTCTCCTAACTGT570
    ProPheGluCysProSerIleGlnIleThrSerIleSerProAsnCys
    105110115120
    CATCAAGAATTAGATGCACATGAAGATGACCTACAGATAAATGACCCA618
    HisGlnGluLeuAspAlaHisGluAspAspLeuGlnIleAsnAspPro
    125130135
    GAACGGGAATTTTTGGAAAGGCCTTCTAGAGATCATCTCTATCTTCCT666
    GluArgGluPheLeuGluArgProSerArgAspHisLeuTyrLeuPro
    140145150
    CTTGAGCCATCCTACCGGGAGTCTTCTCTTAGTCCTAGTCCTGCCAGC714
    LeuGluProSerTyrArgGluSerSerLeuSerProSerProAlaSer
    155160165
    AGCATCTCTTCTAGGAGTTGGTTCTCTGATGCATCTTCTTGTGAATCG762
    SerIleSerSerArgSerTrpPheSerAspAlaSerSerCysGluSer
    170175180
    CTTTCACATATTTATGATGATGTGGACTCAGAGTTGAATGAAGCTGCA810
    LeuSerHisIleTyrAspAspValAspSerGluLeuAsnGluAlaAla
    185190195200
    GCCCGATTTACCCTTGGATCCCCTCTGACTTCTCCTGGTGGCTCTCCA858
    AlaArgPheThrLeuGlySerProLeuThrSerProGlyGlySerPro
    205210215
    GGGGGCTGCCCTGGAGAAGAAACTTGGCATCAACAGTATGGACTTGGA906
    GlyGlyCysProGlyGluGluThrTrpHisGlnGlnTyrGlyLeuGly
    220225230
    CACTCATTATCACCCAGGCAATCTCCTTGCCACTCTCCTAGATCCAGT954
    HisSerLeuSerProArgGlnSerProCysHisSerProArgSerSer
    235240245
    GTCACTGATGAGAATTGGCTGAGCCCCAGGCCAGCCTCAGGACCCTCA1002
    ValThrAspGluAsnTrpLeuSerProArgProAlaSerGlyProSer
    250255260
    TCAAGGCCCACATCCCCCTGTGGGAAACGGAGGCACTCCAGTGCTGAA1050
    SerArgProThrSerProCysGlyLysArgArgHisSerSerAlaGlu
    265270275280
    GTTTGTTATGCTGGGTCCCTTTCACCCCATCACTCACCTGTTCCTTCA1098
    ValCysTyrAlaGlySerLeuSerProHisHisSerProValProSer
    285290295
    CCTGGTCACTCCCCCAGGGGAAGTGTGACAGAAGATACGTGGCTCAAT1146
    ProGlyHisSerProArgGlySerValThrGluAspThrTrpLeuAsn
    300305310
    GCTTCTGTCCATGGTGGGTCAGGCCTTGGCCCTGCAGTTTTTCCATTT1194
    AlaSerValHisGlyGlySerGlyLeuGlyProAlaValPheProPhe
    315320325
    CAGTACTGTGTAGAGACTGACATCCCTCTCAAAACAAGGAAAACTTCT1242
    GlnTyrCysValGluThrAspIleProLeuLysThrArgLysThrSer
    330335340
    GAAGATCAAGCTGCCATACTACCAGGAAAATTAGAGCTGTGTTCAGAT1290
    GluAspGlnAlaAlaIleLeuProGlyLysLeuGluLeuCysSerAsp
    345350355360
    GACCAAGGGAGTTTATCACCAGCCCGGGAGACTTCAATAGATGATGGC1338
    AspGlnGlySerLeuSerProAlaArgGluThrSerIleAspAspGly
    365370375
    CTTGGATCTCAGTATCCTTTAAAGAAAGATTCATGTGGTGATCAGTTT1386
    LeuGlySerGlnTyrProLeuLysLysAspSerCysGlyAspGlnPhe
    380385390
    CTTTCAGTTCCTTCACCCTTTACCTGGAGCAAACCAAAGCCTGGCCAC1434
    LeuSerValProSerProPheThrTrpSerLysProLysProGlyHis
    395400405
    ACCCCTATATTTCGCACATCTTCATTACCTCCACTAGACTGGCCTTTA1482
    ThrProIlePheArgThrSerSerLeuProProLeuAspTrpProLeu
    410415420
    CCAGCTCATTTTGGACAATGTGAACTGAAAATAGAAGTGCAACCTAAA1530
    ProAlaHisPheGlyGlnCysGluLeuLysIleGluValGlnProLys
    425430435440
    ACTCATCATCGAGCCCATTATGAAACTGAAGGTAGCCGAGGGGCAGTA1578
    ThrHisHisArgAlaHisTyrGluThrGluGlySerArgGlyAlaVal
    445450455
    AAAGCATCTACTGGGGGACATCCTGTTGTGAAGCTCCTGGGCTATAAC1626
    LysAlaSerThrGlyGlyHisProValValLysLeuLeuGlyTyrAsn
    460465470
    GAAAAGCCAATAAATCTACAAATGTTTATTGGGACAGCAGATGATCGA1674
    GluLysProIleAsnLeuGlnMetPheIleGlyThrAlaAspAspArg
    475480485
    TATTTACGACCTCATGCATTTTACCAGGTGCATCGAATCACTGGGAAG1722
    TyrLeuArgProHisAlaPheTyrGlnValHisArgIleThrGlyLys
    490495500
    ACAGTCGCTACTGCAAGCCAAGAGATAATAATTGCCAGTACAAAAGTT1770
    ThrValAlaThrAlaSerGlnGluIleIleIleAlaSerThrLysVal
    505510515520
    CTGGAAATTCCACTTCTTCCTGAAAATAATATGTCAGCCAGTATTGAT1818
    LeuGluIleProLeuLeuProGluAsnAsnMetSerAlaSerIleAsp
    525530535
    TGTGCAGGTATTTTGAAACTCCGCAATTCAGATATAGAACTTCGAAAA1866
    CysAlaGlyIleLeuLysLeuArgAsnSerAspIleGluLeuArgLys
    540545550
    GGAGAAACTGATATTGGCAGAAAGAATACTAGAGTACGACTTGTGTTT1914
    GlyGluThrAspIleGlyArgLysAsnThrArgValArgLeuValPhe
    555560565
    CGTGTACACATCCCACAGCCCAGTGGAAAAGTCCTTTCTCTGCAGATA1962
    ArgValHisIleProGlnProSerGlyLysValLeuSerLeuGlnIle
    570575580
    GCCTCTATACCCGTTGAGTGCTCCCAGCGGTCTGCTCAAGAACTTCCT2010
    AlaSerIleProValGluCysSerGlnArgSerAlaGlnGluLeuPro
    585590595600
    CATATTGAGAAGTACAGTATCAACAGTTGTTCTGTAAATGGAGGTCAT2058
    HisIleGluLysTyrSerIleAsnSerCysSerValAsnGlyGlyHis
    605610615
    GAAATGGTTGTGACTGGATCTAATTTTCTTCCAGAATCCAAAATCATT2106
    GluMetValValThrGlySerAsnPheLeuProGluSerLysIleIle
    620625630
    TTTCTTGAAAAAGGACAAGATGGACGACCTCAGTGGGAGGTAGAAGGG2154
    PheLeuGluLysGlyGlnAspGlyArgProGlnTrpGluValGluGly
    635640645
    AAGATAATCAGGGAAAAATGTCAAGGGGCTCACATTGTCCTTGAAGTT2202
    LysIleIleArgGluLysCysGlnGlyAlaHisIleValLeuGluVal
    650655660
    CCTCCATATCATAACCCAGCAGTTACAGCTGCAGTGCAGGTGCACTTT2250
    ProProTyrHisAsnProAlaValThrAlaAlaValGlnValHisPhe
    665670675680
    TATCTTTGCAATGGCAAGAGGAAAAAAAGCCAGTCTCAACGTTTTACT2298



    TyrLeuCysAsnGlyLysArgLysLysSerGlnSerGlnArgPheThr
    685690695
    TATACACCAGTTTTGATGAAGCAAGAACACAGAGAAGAGATTGATTTG2346
    TyrThrProValLeuMetLysGlnGluHisArgGluGluIleAspLeu
    700705710
    TCTTCAGTTCCAACTTTGCCACAGACCTCTCGGCAAACTCTGCTCGGG2394
    SerSerValProThrLeuProGlnThrSerArgGlnThrLeuLeuGly
    715720725
    TCTCAGCCTCCTTCAGCTTCTCCTCCAACAGTTTGATCTCCTCTTCATATTTA2447
    SerGlnProProSerAlaSerProProThrVal
    730735
    TCTTCTTTGGTGGAATACTTGTCCGCCTGGGCCTCCAGGGATTTCAAGTTGTTGGTAACA2507
    ATTTTCAGCTCCTCCTCTAGGTCCCCACATTTACTCTCGGCCACCTCAGCCCTCTCCTCC2567
    GAGCGCTCCAGCTCTCCTTCCAGGATCACCAGCTTCCTGGCCACCTCTTCATATTTGCGG2627
    TCTGAATCCTCAGCGATGTG2647
    (2) INFORMATION FOR SEQ ID NO:10:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 739 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10:
    MetThrThrAlaAsnCysGlyAlaHisAspGluLeuAspPheLysLeu
    151015
    ValPheGlyGluAspGlyAlaProAlaProProProProGlySerArg
    202530
    ProAlaAspLeuGluProAspAspCysAlaSerIleTyrIlePheAsn
    354045
    ValAspProProProSerThrLeuThrThrProLeuCysLeuProHis
    505560
    HisGlyLeuProSerHisSerSerValLeuSerProSerPheGlnLeu
    65707580
    GlnSerHisLysAsnTyrGluGlyThrCysGluIleProGluSerLys
    859095
    TyrSerProLeuGlyGlyProLysProPheGluCysProSerIleGln
    100105110
    IleThrSerIleSerProAsnCysHisGlnGluLeuAspAlaHisGlu
    115120125
    AspAspLeuGlnIleAsnAspProGluArgGluPheLeuGluArgPro
    130135140
    SerArgAspHisLeuTyrLeuProLeuGluProSerTyrArgGluSer
    145150155160
    SerLeuSerProSerProAlaSerSerIleSerSerArgSerTrpPhe
    165170175
    SerAspAlaSerSerCysGluSerLeuSerHisIleTyrAspAspVal
    180185190
    AspSerGluLeuAsnGluAlaAlaAlaArgPheThrLeuGlySerPro
    195200205
    LeuThrSerProGlyGlySerProGlyGlyCysProGlyGluGluThr
    210215220
    TrpHisGlnGlnTyrGlyLeuGlyHisSerLeuSerProArgGlnSer
    225230235240
    ProCysHisSerProArgSerSerValThrAspGluAsnTrpLeuSer
    245250255
    ProArgProAlaSerGlyProSerSerArgProThrSerProCysGly
    260265270
    LysArgArgHisSerSerAlaGluValCysTyrAlaGlySerLeuSer
    275280285
    ProHisHisSerProValProSerProGlyHisSerProArgGlySer
    290295300
    ValThrGluAspThrTrpLeuAsnAlaSerValHisGlyGlySerGly
    305310315320
    LeuGlyProAlaValPheProPheGlnTyrCysValGluThrAspIle
    325330335
    ProLeuLysThrArgLysThrSerGluAspGlnAlaAlaIleLeuPro
    340345350
    GlyLysLeuGluLeuCysSerAspAspGlnGlySerLeuSerProAla
    355360365
    ArgGluThrSerIleAspAspGlyLeuGlySerGlnTyrProLeuLys
    370375380
    LysAspSerCysGlyAspGlnPheLeuSerValProSerProPheThr
    385390395400
    TrpSerLysProLysProGlyHisThrProIlePheArgThrSerSer
    405410415
    LeuProProLeuAspTrpProLeuProAlaHisPheGlyGlnCysGlu
    420425430
    LeuLysIleGluValGlnProLysThrHisHisArgAlaHisTyrGlu
    435440445
    ThrGluGlySerArgGlyAlaValLysAlaSerThrGlyGlyHisPro
    450455460
    ValValLysLeuLeuGlyTyrAsnGluLysProIleAsnLeuGlnMet
    465470475480
    PheIleGlyThrAlaAspAspArgTyrLeuArgProHisAlaPheTyr
    485490495
    GlnValHisArgIleThrGlyLysThrValAlaThrAlaSerGlnGlu
    500505510
    IleIleIleAlaSerThrLysValLeuGluIleProLeuLeuProGlu
    515520525
    AsnAsnMetSerAlaSerIleAspCysAlaGlyIleLeuLysLeuArg
    530535540
    AsnSerAspIleGluLeuArgLysGlyGluThrAspIleGlyArgLys
    545550555560
    AsnThrArgValArgLeuValPheArgValHisIleProGlnProSer
    565570575
    GlyLysValLeuSerLeuGlnIleAlaSerIleProValGluCysSer
    580585590
    GlnArgSerAlaGlnGluLeuProHisIleGluLysTyrSerIleAsn
    595600605
    SerCysSerValAsnGlyGlyHisGluMetValValThrGlySerAsn
    610615620
    PheLeuProGluSerLysIleIlePheLeuGluLysGlyGlnAspGly
    625630635640
    ArgProGlnTrpGluValGluGlyLysIleIleArgGluLysCysGln
    645650655
    GlyAlaHisIleValLeuGluValProProTyrHisAsnProAlaVal
    660665670
    ThrAlaAlaValGlnValHisPheTyrLeuCysAsnGlyLysArgLys
    675680685
    LysSerGlnSerGlnArgPheThrTyrThrProValLeuMetLysGln
    690695700
    GluHisArgGluGluIleAspLeuSerSerValProThrLeuProGln
    705710715720
    ThrSerArgGlnThrLeuLeuGlySerGlnProProSerAlaSerPro
    725730735
    ProThrVal
    (2) INFORMATION FOR SEQ ID NO:11:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 3969 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: double
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (ix) FEATURE:
    (A) NAME/KEY: CDS
    (B) LOCATION: 211..3414
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:11:
    CGGCTGCGGTTCCTGGTGCTGCTCGGCGCGCGGCCAGCTTTCGGAACGGAACGCTCGGCG60
    TCGCGGGCCCCGCCCGGAAAGTTTGCCGTGGAGTCGCGACCTCTTGGCCCGCGCGGCCCG120
    GCATGAAGCGGCGTTGAGGAGCTGCTGCCGCCGCTTGCCGCTGCCGCCGCCGCCGCCTGA180
    GGAGGAGCTGCAGCACCCTGGGCCACGCCGATGACTACTGCAAACTGTGGCGCC234
    MetThrThrAlaAsnCysGlyAla
    740745
    CACGACGAGCTCGACTTCAAACTCGTCTTTGGCGAGGACGGGGCGCCG282
    HisAspGluLeuAspPheLysLeuValPheGlyGluAspGlyAlaPro
    750755760
    GCGCCGCCGCCCCCGGGCTCGCGGCCTGCAGATCTTGAGCCAGATGAT330
    AlaProProProProGlySerArgProAlaAspLeuGluProAspAsp
    765770775
    TGTGCATCCATTTACATCTTTAATGTAGATCCACCTCCATCTACTTTA378
    CysAlaSerIleTyrIlePheAsnValAspProProProSerThrLeu
    780785790795
    ACCACACCACTTTGCTTACCACATCATGGATTACCGTCTCACTCTTCT426
    ThrThrProLeuCysLeuProHisHisGlyLeuProSerHisSerSer
    800805810
    GTTTTGTCACCATCGTTTCAGCTCCAAAGTCACAAAAACTATGAAGGA474
    ValLeuSerProSerPheGlnLeuGlnSerHisLysAsnTyrGluGly
    815820825
    ACTTGTGAGATTCCTGAATCTAAATATAGCCCATTAGGTGGTCCCAAA522
    ThrCysGluIleProGluSerLysTyrSerProLeuGlyGlyProLys
    830835840
    CCCTTTGAGTGCCCAAGTATTCAAATTACATCTATCTCTCCTAACTGT570
    ProPheGluCysProSerIleGlnIleThrSerIleSerProAsnCys
    845850855
    CATCAAGAATTAGATGCACATGAAGATGACCTACAGATAAATGACCCA618
    HisGlnGluLeuAspAlaHisGluAspAspLeuGlnIleAsnAspPro
    860865870875
    GAACGGGAATTTTTGGAAAGGCCTTCTAGAGATCATCTCTATCTTCCT666
    GluArgGluPheLeuGluArgProSerArgAspHisLeuTyrLeuPro
    880885890
    CTTGAGCCATCCTACCGGGAGTCTTCTCTTAGTCCTAGTCCTGCCAGC714
    LeuGluProSerTyrArgGluSerSerLeuSerProSerProAlaSer
    895900905
    AGCATCTCTTCTAGGAGTTGGTTCTCTGATGCATCTTCTTGTGAATCG762
    SerIleSerSerArgSerTrpPheSerAspAlaSerSerCysGluSer
    910915920
    CTTTCACATATTTATGATGATGTGGACTCAGAGTTGAATGAAGCTGCA810
    LeuSerHisIleTyrAspAspValAspSerGluLeuAsnGluAlaAla
    925930935
    GCCCGATTTACCCTTGGATCCCCTCTGACTTCTCCTGGTGGCTCTCCA858
    AlaArgPheThrLeuGlySerProLeuThrSerProGlyGlySerPro
    940945950955
    GGGGGCTGCCCTGGAGAAGAAACTTGGCATCAACAGTATGGACTTGGA906
    GlyGlyCysProGlyGluGluThrTrpHisGlnGlnTyrGlyLeuGly
    960965970
    CACTCATTATCACCCAGGCAATCTCCTTGCCACTCTCCTAGATCCAGT954
    HisSerLeuSerProArgGlnSerProCysHisSerProArgSerSer
    975980985
    GTCACTGATGAGAATTGGCTGAGCCCCAGGCCAGCCTCAGGACCCTCA1002
    ValThrAspGluAsnTrpLeuSerProArgProAlaSerGlyProSer
    9909951000
    TCAAGGCCCACATCCCCCTGTGGGAAACGGAGGCACTCCAGTGCTGAA1050
    SerArgProThrSerProCysGlyLysArgArgHisSerSerAlaGlu
    100510101015
    GTTTGTTATGCTGGGTCCCTTTCACCCCATCACTCACCTGTTCCTTCA1098
    ValCysTyrAlaGlySerLeuSerProHisHisSerProValProSer
    1020102510301035
    CCTGGTCACTCCCCCAGGGGAAGTGTGACAGAAGATACGTGGCTCAAT1146
    ProGlyHisSerProArgGlySerValThrGluAspThrTrpLeuAsn
    104010451050
    GCTTCTGTCCATGGTGGGTCAGGCCTTGGCCCTGCAGTTTTTCCATTT1194
    AlaSerValHisGlyGlySerGlyLeuGlyProAlaValPheProPhe
    105510601065
    CAGTACTGTGTAGAGACTGACATCCCTCTCAAAACAAGGAAAACTTCT1242
    GlnTyrCysValGluThrAspIleProLeuLysThrArgLysThrSer
    107010751080
    GAAGATCAAGCTGCCATACTACCAGGAAAATTAGAGCTGTGTTCAGAT1290
    GluAspGlnAlaAlaIleLeuProGlyLysLeuGluLeuCysSerAsp
    108510901095
    GACCAAGGGAGTTTATCACCAGCCCGGGAGACTTCAATAGATGATGGC1338
    AspGlnGlySerLeuSerProAlaArgGluThrSerIleAspAspGly
    1100110511101115
    CTTGGATCTCAGTATCCTTTAAAGAAAGATTCATGTGGTGATCAGTTT1386
    LeuGlySerGlnTyrProLeuLysLysAspSerCysGlyAspGlnPhe
    112011251130
    CTTTCAGTTCCTTCACCCTTTACCTGGAGCAAACCAAAGCCTGGCCAC1434
    LeuSerValProSerProPheThrTrpSerLysProLysProGlyHis
    113511401145
    ACCCCTATATTTCGCACATCTTCATTACCTCCACTAGACTGGCCTTTA1482
    ThrProIlePheArgThrSerSerLeuProProLeuAspTrpProLeu
    115011551160
    CCAGCTCATTTTGGACAATGTGAACTGAAAATAGAAGTGCAACCTAAA1530
    ProAlaHisPheGlyGlnCysGluLeuLysIleGluValGlnProLys
    116511701175
    ACTCATCATCGAGCCCATTATGAAACTGAAGGTAGCCGAGGGGCAGTA1578
    ThrHisHisArgAlaHisTyrGluThrGluGlySerArgGlyAlaVal
    1180118511901195
    AAAGCATCTACTGGGGGACATCCTGTTGTGAAGCTCCTGGGCTATAAC1626
    LysAlaSerThrGlyGlyHisProValValLysLeuLeuGlyTyrAsn
    120012051210
    GAAAAGCCAATAAATCTACAAATGTTTATTGGGACAGCAGATGATCGA1674
    GluLysProIleAsnLeuGlnMetPheIleGlyThrAlaAspAspArg
    121512201225
    TATTTACGACCTCATGCATTTTACCAGGTGCATCGAATCACTGGGAAG1722
    TyrLeuArgProHisAlaPheTyrGlnValHisArgIleThrGlyLys
    123012351240
    ACAGTCGCTACTGCAAGCCAAGAGATAATAATTGCCAGTACAAAAGTT1770
    ThrValAlaThrAlaSerGlnGluIleIleIleAlaSerThrLysVal
    124512501255
    CTGGAAATTCCACTTCTTCCTGAAAATAATATGTCAGCCAGTATTGAT1818
    LeuGluIleProLeuLeuProGluAsnAsnMetSerAlaSerIleAsp
    1260126512701275
    TGTGCAGGTATTTTGAAACTCCGCAATTCAGATATAGAACTTCGAAAA1866
    CysAlaGlyIleLeuLysLeuArgAsnSerAspIleGluLeuArgLys
    128012851290
    GGAGAAACTGATATTGGCAGAAAGAATACTAGAGTACGACTTGTGTTT1914
    GlyGluThrAspIleGlyArgLysAsnThrArgValArgLeuValPhe
    129513001305
    CGTGTACACATCCCACAGCCCAGTGGAAAAGTCCTTTCTCTGCAGATA1962
    ArgValHisIleProGlnProSerGlyLysValLeuSerLeuGlnIle
    131013151320
    GCCTCTATACCCGTTGAGTGCTCCCAGCGGTCTGCTCAAGAACTTCCT2010
    AlaSerIleProValGluCysSerGlnArgSerAlaGlnGluLeuPro
    132513301335
    CATATTGAGAAGTACAGTATCAACAGTTGTTCTGTAAATGGAGGTCAT2058
    HisIleGluLysTyrSerIleAsnSerCysSerValAsnGlyGlyHis
    1340134513501355
    GAAATGGTTGTGACTGGATCTAATTTTCTTCCAGAATCCAAAATCATT2106
    GluMetValValThrGlySerAsnPheLeuProGluSerLysIleIle
    136013651370
    TTTCTTGAAAAAGGACAAGATGGACGACCTCAGTGGGAGGTAGAAGGG2154
    PheLeuGluLysGlyGlnAspGlyArgProGlnTrpGluValGluGly
    137513801385
    AAGATAATCAGGGAAAAATGTCAAGGGGCTCACATTGTCCTTGAAGTT2202
    LysIleIleArgGluLysCysGlnGlyAlaHisIleValLeuGluVal
    139013951400



    CCTCCATATCATAACCCAGCAGTTACAGCTGCAGTGCAGGTGCACTTT2250
    ProProTyrHisAsnProAlaValThrAlaAlaValGlnValHisPhe
    140514101415
    TATCTTTGCAATGGCAAGAGGAAAAAAAGCCAGTCTCAACGTTTTACT2298
    TyrLeuCysAsnGlyLysArgLysLysSerGlnSerGlnArgPheThr
    1420142514301435
    TATACACCAGTTTTGATGAAGCAAGAACACAGAGAAGAGATTGATTTG2346
    TyrThrProValLeuMetLysGlnGluHisArgGluGluIleAspLeu
    144014451450
    TCTTCAGTTCCATCTTTGCCTGTGCCTCATCCTGCTCAGACCCAGAGG2394
    SerSerValProSerLeuProValProHisProAlaGlnThrGlnArg
    145514601465
    CCTTCCTCTGATTCAGGGTGTTCACATGACAGTGTACTGTCAGGACAG2442
    ProSerSerAspSerGlyCysSerHisAspSerValLeuSerGlyGln
    147014751480
    AGAAGTTTGATTTGCTCCATCCCACAAACATATGCATCCATGGTGACC2490
    ArgSerLeuIleCysSerIleProGlnThrTyrAlaSerMetValThr
    148514901495
    TCATCCCATCTGCCACAGTTGCAGTGTAGAGATGAGAGTGTTAGTAAA2538
    SerSerHisLeuProGlnLeuGlnCysArgAspGluSerValSerLys
    1500150515101515
    GAACAGCATATGATTCCTTCTCCAATTGTACACCAGCCTTTTCAAGTC2586
    GluGlnHisMetIleProSerProIleValHisGlnProPheGlnVal
    152015251530
    ACACCAACACCTCCTGTGGGGTCTTCCTATCAGCCTATGCAAACTAAT2634
    ThrProThrProProValGlySerSerTyrGlnProMetGlnThrAsn
    153515401545
    GTTGTGTACAATGGACCAACTTGTCTTCCTATTAATGCTGCCTCTAGT2682
    ValValTyrAsnGlyProThrCysLeuProIleAsnAlaAlaSerSer
    155015551560
    CAAGAATTTGATTCAGTTTTGTTTCAGCAGGATGCAACTCTTTCTGGT2730
    GlnGluPheAspSerValLeuPheGlnGlnAspAlaThrLeuSerGly
    156515701575
    TTAGTGAATCTTGGCTGTCAACCACTGTCATCCATACCATTTCATTCT2778
    LeuValAsnLeuGlyCysGlnProLeuSerSerIleProPheHisSer
    1580158515901595
    TCAAATTCAGGCTCAACAGGACATCTCTTAGCCCATACACCTCATTCT2826
    SerAsnSerGlySerThrGlyHisLeuLeuAlaHisThrProHisSer
    160016051610
    GTGCATACCCTGCCTCATCTGCAATCAATGGGATATCATTGTTCAAAT2874
    ValHisThrLeuProHisLeuGlnSerMetGlyTyrHisCysSerAsn
    161516201625
    ACAGGACAAAGATCTCTTTCTTCTCCAGTGGCTGACCAGATTACAGGT2922
    ThrGlyGlnArgSerLeuSerSerProValAlaAspGlnIleThrGly
    163016351640
    CAGCCTTCGTCTCAGTTACAACCTATTACATATGGTCCTTCACATTCA2970
    GlnProSerSerGlnLeuGlnProIleThrTyrGlyProSerHisSer
    164516501655
    GGGTCTGCTACAACAGCTTCCCCAGCAGCTTCTCATCCCTTGGCTAGT3018
    GlySerAlaThrThrAlaSerProAlaAlaSerHisProLeuAlaSer
    1660166516701675
    TCACCGCTTTCTGGGCCACCATCTCCTCAGCTTCAGCCTATGCCTTAC3066
    SerProLeuSerGlyProProSerProGlnLeuGlnProMetProTyr
    168016851690
    CAATCTCCTAGCTCAGGAACTGCCTCATCACCGTCTCCAGCCACCAGA3114
    GlnSerProSerSerGlyThrAlaSerSerProSerProAlaThrArg
    169517001705
    ATGCATTCTGGACAGCACTCAACTCAAGCACAAAGTACGGGCCAGGGG3162
    MetHisSerGlyGlnHisSerThrGlnAlaGlnSerThrGlyGlnGly
    171017151720
    GGTCTTTCTGCACCTTCATCCTTAATATGTCACAGTTTGTGTGATCCA3210
    GlyLeuSerAlaProSerSerLeuIleCysHisSerLeuCysAspPro
    172517301735
    GCGTCATTTCCACCTGATGGGGCAACTGTGAGCATTAAACCTGAACCA3258
    AlaSerPheProProAspGlyAlaThrValSerIleLysProGluPro
    1740174517501755
    GAAGATCGAGAGCCTAACTTTGCAACCATTGGTCTGCAGGACATCACT3306
    GluAspArgGluProAsnPheAlaThrIleGlyLeuGlnAspIleThr
    176017651770
    TTAGATGATGACCAATTTATATCTGACTTGGAACACCAGCCATCAGGT3354
    LeuAspAspAspGlnPheIleSerAspLeuGluHisGlnProSerGly
    177517801785
    TCAGCAGAGAAATGGCCTAACCACAGTGTGCTCTCATGTCCAGCTCCT3402
    SerAlaGluLysTrpProAsnHisSerValLeuSerCysProAlaPro
    179017951800
    TTCTGGAGAATCTAGAGGTGAACGAGATAATTGGGAGAGACATGTCCCAGAT3454
    PheTrpArgIle
    1805
    TTCTGTTTCCCAAGGAGCAGGGGTGAGCAGGCAGGCTCCCCTCCCGAGTCCTGAGTCCCT3514
    GGATTTAGGAAGATCTGATGGGCTCTAACAGTGCTTACTGCAGCCTTGTGTCCACCACCA3574
    ACTTCTCAGCATGTTTCTCTCCTTGGACCTTGGGTTTCCAACTCTGCAGCCTTCAGGTCT3634
    GGGGCCAGGAGTGGGACCCACCATTTGTGGGGAAAGTAGCATTCCTCCACCTCAGGCCTT3694
    GGGTAGATTTGGCAAAAGAACAGGAGCAGCATAGGCTGTTTGAGCTTTGGGGAAATGAAC3754
    TTTGCTTTTTATATTTAACTAGGATACTTTTATATGATGGGTGCTTTGAGTGTGAATGCA3814
    GCAGGCTCTCTTGTTTCCGAGGTGCTGCTTTTGCAGGTGACCTGGTTACTTAGCTAGGAT3874
    TGGTGATTTGTACTGCTTTATGGTCATTTGAAGGGCCCTTTAGTTTTTATGATAATTTTT3934
    AAAATAGGAACTTTTGATAAGACCTTCTAGAAGCC3969
    (2) INFORMATION FOR SEQ ID NO:12:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 1068 amino acids
    (B) TYPE: amino acid
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: protein
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12:
    MetThrThrAlaAsnCysGlyAlaHisAspGluLeuAspPheLysLeu
    151015
    ValPheGlyGluAspGlyAlaProAlaProProProProGlySerArg
    202530
    ProAlaAspLeuGluProAspAspCysAlaSerIleTyrIlePheAsn
    354045
    ValAspProProProSerThrLeuThrThrProLeuCysLeuProHis
    505560
    HisGlyLeuProSerHisSerSerValLeuSerProSerPheGlnLeu
    65707580
    GlnSerHisLysAsnTyrGluGlyThrCysGluIleProGluSerLys
    859095
    TyrSerProLeuGlyGlyProLysProPheGluCysProSerIleGln
    100105110
    IleThrSerIleSerProAsnCysHisGlnGluLeuAspAlaHisGlu
    115120125
    AspAspLeuGlnIleAsnAspProGluArgGluPheLeuGluArgPro
    130135140
    SerArgAspHisLeuTyrLeuProLeuGluProSerTyrArgGluSer
    145150155160
    SerLeuSerProSerProAlaSerSerIleSerSerArgSerTrpPhe
    165170175
    SerAspAlaSerSerCysGluSerLeuSerHisIleTyrAspAspVal
    180185190
    AspSerGluLeuAsnGluAlaAlaAlaArgPheThrLeuGlySerPro
    195200205
    LeuThrSerProGlyGlySerProGlyGlyCysProGlyGluGluThr
    210215220
    TrpHisGlnGlnTyrGlyLeuGlyHisSerLeuSerProArgGlnSer
    225230235240
    ProCysHisSerProArgSerSerValThrAspGluAsnTrpLeuSer
    245250255
    ProArgProAlaSerGlyProSerSerArgProThrSerProCysGly
    260265270
    LysArgArgHisSerSerAlaGluValCysTyrAlaGlySerLeuSer
    275280285
    ProHisHisSerProValProSerProGlyHisSerProArgGlySer
    290295300
    ValThrGluAspThrTrpLeuAsnAlaSerValHisGlyGlySerGly
    305310315320
    LeuGlyProAlaValPheProPheGlnTyrCysValGluThrAspIle
    325330335
    ProLeuLysThrArgLysThrSerGluAspGlnAlaAlaIleLeuPro
    340345350
    GlyLysLeuGluLeuCysSerAspAspGlnGlySerLeuSerProAla
    355360365
    ArgGluThrSerIleAspAspGlyLeuGlySerGlnTyrProLeuLys
    370375380
    LysAspSerCysGlyAspGlnPheLeuSerValProSerProPheThr
    385390395400
    TrpSerLysProLysProGlyHisThrProIlePheArgThrSerSer
    405410415
    LeuProProLeuAspTrpProLeuProAlaHisPheGlyGlnCysGlu
    420425430
    LeuLysIleGluValGlnProLysThrHisHisArgAlaHisTyrGlu
    435440445
    ThrGluGlySerArgGlyAlaValLysAlaSerThrGlyGlyHisPro
    450455460
    ValValLysLeuLeuGlyTyrAsnGluLysProIleAsnLeuGlnMet
    465470475480
    PheIleGlyThrAlaAspAspArgTyrLeuArgProHisAlaPheTyr
    485490495
    GlnValHisArgIleThrGlyLysThrValAlaThrAlaSerGlnGlu
    500505510
    IleIleIleAlaSerThrLysValLeuGluIleProLeuLeuProGlu
    515520525
    AsnAsnMetSerAlaSerIleAspCysAlaGlyIleLeuLysLeuArg
    530535540
    AsnSerAspIleGluLeuArgLysGlyGluThrAspIleGlyArgLys
    545550555560
    AsnThrArgValArgLeuValPheArgValHisIleProGlnProSer
    565570575
    GlyLysValLeuSerLeuGlnIleAlaSerIleProValGluCysSer
    580585590
    GlnArgSerAlaGlnGluLeuProHisIleGluLysTyrSerIleAsn
    595600605
    SerCysSerValAsnGlyGlyHisGluMetValValThrGlySerAsn
    610615620
    PheLeuProGluSerLysIleIlePheLeuGluLysGlyGlnAspGly
    625630635640
    ArgProGlnTrpGluValGluGlyLysIleIleArgGluLysCysGln
    645650655
    GlyAlaHisIleValLeuGluValProProTyrHisAsnProAlaVal
    660665670
    ThrAlaAlaValGlnValHisPheTyrLeuCysAsnGlyLysArgLys
    675680685
    LysSerGlnSerGlnArgPheThrTyrThrProValLeuMetLysGln
    690695700
    GluHisArgGluGluIleAspLeuSerSerValProSerLeuProVal
    705710715720
    ProHisProAlaGlnThrGlnArgProSerSerAspSerGlyCysSer
    725730735
    HisAspSerValLeuSerGlyGlnArgSerLeuIleCysSerIlePro
    740745750
    GlnThrTyrAlaSerMetValThrSerSerHisLeuProGlnLeuGln
    755760765
    CysArgAspGluSerValSerLysGluGlnHisMetIleProSerPro
    770775780
    IleValHisGlnProPheGlnValThrProThrProProValGlySer
    785790795800
    SerTyrGlnProMetGlnThrAsnValValTyrAsnGlyProThrCys
    805810815
    LeuProIleAsnAlaAlaSerSerGlnGluPheAspSerValLeuPhe
    820825830
    GlnGlnAspAlaThrLeuSerGlyLeuValAsnLeuGlyCysGlnPro
    835840845
    LeuSerSerIleProPheHisSerSerAsnSerGlySerThrGlyHis
    850855860
    LeuLeuAlaHisThrProHisSerValHisThrLeuProHisLeuGln
    865870875880
    SerMetGlyTyrHisCysSerAsnThrGlyGlnArgSerLeuSerSer
    885890895
    ProValAlaAspGlnIleThrGlyGlnProSerSerGlnLeuGlnPro
    900905910
    IleThrTyrGlyProSerHisSerGlySerAlaThrThrAlaSerPro
    915920925
    AlaAlaSerHisProLeuAlaSerSerProLeuSerGlyProProSer
    930935940
    ProGlnLeuGlnProMetProTyrGlnSerProSerSerGlyThrAla
    945950955960
    SerSerProSerProAlaThrArgMetHisSerGlyGlnHisSerThr
    965970975
    GlnAlaGlnSerThrGlyGlnGlyGlyLeuSerAlaProSerSerLeu
    980985990
    IleCysHisSerLeuCysAspProAlaSerPheProProAspGlyAla
    99510001005
    ThrValSerIleLysProGluProGluAspArgGluProAsnPheAla
    101010151020
    ThrIleGlyLeuGlnAspIleThrLeuAspAspAspGlnPheIleSer
    1025103010351040
    AspLeuGluHisGlnProSerGlySerAlaGluLysTrpProAsnHis
    104510501055
    SerValLeuSerCysProAlaProPheTrpArgIle
    10601065
    (2) INFORMATION FOR SEQ ID NO:13:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 27 amino acids
    (B) TYPE: amino acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13:
    AspIleGluLeuArgLysGlyGluThrAspIleGlyArgLysAsnThr
    151015
    ArgValArgLeuValPheArgValHisXaaPro
    2025
    (2) INFORMATION FOR SEQ ID NO:14:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 13 amino acids
    (B) TYPE: amino acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: peptide
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14:
    ProXaaGluCysSerGlnArgSerAlaXaaGluLeuPro
    1510
    (2) INFORMATION FOR SEQ ID NO:15:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single



    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:15:
    GGAAAATTTT10
    (2) INFORMATION FOR SEQ ID NO:16:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 10 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:16:
    GGAAAAACTG10
    (2) INFORMATION FOR SEQ ID NO:17:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 23 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:17:
    TACATTGGAAAATTTTATTACAC23
    (2) INFORMATION FOR SEQ ID NO:18:
    (i) SEQUENCE CHARACTERISTICS:
    (A) LENGTH: 30 base pairs
    (B) TYPE: nucleic acid
    (C) STRANDEDNESS: single
    (D) TOPOLOGY: linear
    (ii) MOLECULE TYPE: cDNA
    (xi) SEQUENCE DESCRIPTION: SEQ ID NO:18:
    GGAGGAAAAACTGTTTCATACAGAAGGCGT30
    __________________________________________________________________________


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